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251461502 | pes2o/s2orc | v3-fos-license | Molecular diversity of the base-promoted reaction of phenacylmalononitriles with dialkyl but-2-ynedioates
In the presence of tetrabutylammonium bromide (TBAB), the cycloaddition reaction of phenacylmalononitriles with dialkyl but-2-ynedioates in acetonitrile at room temperature resulted in 3,3-dicyano-5-hydroxy-5-arylcyclopent-1-ene-1,2-dicarboxylates in high yields. More importantly, the DABCO-promoted domino reaction of two molecules of each phenacylmalononitrile and dialkyl but-2-ynedioate in acetonitrile at room temperature afforded unique multifunctionalized carboxamide-bridged dicyclopentenes in moderate to good yields and with high diastereoselectivity.
Results and Discussion
Initially, the reaction conditions were briefly optimized by using p-methylphenacylmalononitrile (1a) and diethyl but-2ynedioate (2a) as standard according to the previous reported work [31]. In the presence of potassium carbonate and tetra-butylammonium chloride (TBAC), the reaction in acetonitrile at room temperature gave diethyl 3,3-dicyano-5-hydroxy-5-(pmethylphenyl)cyclopent-1-ene-1,2-dicarboxylate (3a) in moderate yield (Table 1). In the presence of tetrabutylammonium bromide (TBAB), the reaction in acetonitrile afforded product 3a in 85% yield. The reaction in DCM gave the product 3a in 65% yield. However, no product 3a was obtained when the reaction was carried out in toluene at room temperature or in acetonitrile at 0 °C. The yield of 3a remained the same when the reaction was carried out at elevated temperature. At last, using of higher loading of TBAB and prolonging the reaction time could not increase the yield of the product 3a. Therefore, the functionalized 5-hydroxy-cyclopent-1-ene derivatives can be conveniently prepared in satisfactory yield in very simple reaction conditions. It should be pointed that a similar triethylamine-promoted multicomponent reaction of phenacyl bromide, malononitrile, dialkyl but-2-ynedioate, and triphenylphosphine has been already reported, in which diethyl 3-phenyl-5,5dicyanocyclopent-2-ene-1,2-dicarboxylates were produced by further elimination of a hydroxy group [30]. In the present reaction, the hydroxy group is still remained in the product 3a. This result might be due to the weak basic system and the milder conditions. An attempt to develop a three-component reaction by directly using the phenacyl bromide and malononitrile to replace the previously prepared phenacylmalononitrile in the reaction was not successful. The reaction gave a very complex mixture of products. Under the optimized reaction conditions (Table 1, entry 3), the scope of the reaction was investigated by using various substrates and the results are summarized in Table 2. It can be seen that all reactions proceeded smoothly to give the expected functionalized 5-hydroxycyclopentenes 3a-l in good to excellent yields. The phenacylmalononitriles with electron-donating groups usually gave higher yields than that of substrates bearing an electron-donating chloro, bromo and nitro group. Both diethyl and dimethyl but-2-ynedioates can be successfully employed in the reaction. Because there is only one chiral carbon atom in the molecule, there are no diastereoisomers in the obtained products 3a-l. The chemical structures of compounds 3a-l were fully characterized by IR, HRMS, 1 H and 13 C NMR spectra. As for an example, the 1 H NMR spectrum of compound 3i displayed a singlet at 3.52 ppm for the hydroxy group and two singlets at 3.24, 3.96 ppm with J = 14.8 Hz for the two diastereotopic protons of the cyclic methylene unit. The single crystal structure of compound 3k was successfully determined by X-ray diffraction analysis ( Figure 1). From Figure 1, it can be seen that the C-C double bond is connected to two methoxycarbonyl groups. Though one hydroxy group exists on the reactive allyl position and benzyl position, it still is present in the molecule and did not give the cyclopentadiene by further elimination of water. In order to obtain the corresponding products with elimination of water, alternative conditions for the base-promoted reaction of phenacylmalononitrile and dialkyl but-2-ynedioates were tested. After carefully examining the reaction conditions, we found that the reaction of phenacylmalononitriles and dialkyl but-2-ynedioates in acetonitrile at room temperature gave the unexpected products 4a-k in moderate to good yields in the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO) as base promoter and the results are summarized in Table 3. It can be seen that the products 4a-k contain two scaffolds of each phenacylmalononitrile and dialkyl but-2-ynedioate. Thus, a quasi-four-component reaction led to the final product. There are two chiral carbon atoms in the molecules, thus two diastereoisomers would be formed in the reaction. However, the 1 H and 13 C NMR spectra gave only one set of resonances for the characteristic groups, which clearly indicated that only one diastereoisomer was actually produced in the reaction. The chemical structures of the compounds 4a-k were established by various spectroscopy methods. Additionally, the single crystal structures of compounds 4a and 4c were successfully determined ( Figure 2 and Figure 3). From the two figures, it can be seen that a cyclopentadiene moiety is connected to a cyclopent-1-ene moiety by a carboxamide unit (CONH). Although one hydroxy group is eliminated to give the cyclopentadiene ring, another hydroxy group is still present in the cyclopent-1-ene ring. Additionally, in the cyclopent-1-ene ring, the aryl group and the cyano group are in cis-orientation, while the hydroxy group and carboxamide group exist on the other side of the ring. On the basis of the NMR spectra and the single crystal structures, it can be concluded that all the obtained products 4a-k have this kind of relative configuration.
For explaining the formation of the two kinds of the compounds, a plausible reaction mechanism was proposed on the basis of the previous works [30][31][32][33][34][35] and the present experimental results (Scheme 2). As described in the previous work, TBAF can act as an effective base to catalyze the C-C bond formation via a Michael addition of active methylene groups [31]. Therefore, in the presence of TBAB, the bromide assisted with the deprotonation of the phenacylmalononitrile to give a carbanion intermediate A. Secondly, the nucleophilic addition of carbanion A to electron-deficient alkyne resulted in adduct B. Thirdly, the intramolecular addition of the carbanion to the carbonyl group afforded species C, which in turn converted to the final product 3 by the protonation of the species C. The protonated species 5 could be successfully isolated in 12% yield after six hours when the reaction was carried out in weak basic solution (Supporting Information File 1) and its single crystal structure was determined by X-ray diffraction (Figure 4). In order to shed light on the proposed reaction mechanism, some control experiments were carried out. After finishing the reaction of phenacylmalononitrile and diethyl but-2-ynedioate in the presence of TBAB under the standard reaction conditions, DABCO was directly added to the reaction system (Scheme 3).
The further reaction at room temperature for 24 hours afforded the expected product 4d in 45% yield. This result clearly showed that the initially formed functionalized cyclopentene 3b could be smoothly transferred to the carboxamide-bridged dicyclopentene 4d, which also strongly supported the above proposed reaction mechanism.
Conclusion
In summary, we have investigated the cycloaddition of phenacylmalononitriles and dialkyl but-2-ynedioates under different reaction conditions and identified convenient synthetic protocols for the synthesis of functionalized cyclopent-2-ene and complex dicyclopentene derivatives in satisfactory yields. The stereochemistry of the reactions was clearly elucidated and a rational reaction mechanism was proposed. The reactions have the advantages of using readily available reagents, mild reaction conditions, good yields, and high diastereoselectivity, and have potential synthetic applications in organic and medicinal chemistry. Experimental 1. General procedure for the preparation of functionalized cyclopent-2-enes 3a-l: To a round-bottomed flask was added phenacylmalononitrile (0.5 mmol), dialkyl but-2-ynedioate (0.6 mmol), tetrabutylammonium bromide (0.25 mmol), and acetonitrile (5.0 mL). The solution was stirred at room temperature for twelve hours. After removing the solvent by rotatory evaporation at reduced pressure, the residue was subjected to column chromatography with a mixture of ethyl acetate, petroleum ether and methylene dichloride 1:9:3 (v/v/v) as eluent to give the pure product for analysis. 2. General procedure for the preparation of functionalized carboxamide-bridged dicyclopentenes 4a-k: To a roundbottomed flask was added phenacylmalononitrile (0.5 mmol), dialkyl but-2-ynedioate (0.6 mmol), DABCO (1.0 mmol), and acetonitrile (5.0 mL). The solution was stirred at room temperature for 24 hours. After removing the solvent by rotatory evaporation at reduced pressure, the residue was subjected to column chromatography with a mixture of ethyl acetate and petroleum ether 1:3 (v/v) as eluent to give the pure product for analysis.
Funding
This work was financially supported by National Natural Science Foundation of China (Nos. 21572196, 21871227). | 2022-08-10T15:21:48.227Z | 2022-08-08T00:00:00.000 | {
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201019355 | pes2o/s2orc | v3-fos-license | Prevalence and Severity of Tinnitus in Otosclerosis: Preliminary Findings from Validated Questionnaires
In addition to progressive hearing loss, subjective tinnitus is one of the primary symptoms of the otosclerosis development [3-6]. The pathophysiology behind the tinnitus has not been clearly explained, despite ongoing scientific research. The “exchange hypothesis” of tinnitus formation in otosclerosis assumes that the newly formed bone tissue, because of its rich vascularization, leads to pulsatile tinnitus [7]. According to Ismi et al. [6], tinnitus may be a consequence of the reduction of the inner ear fluid vibration or the production of toxic otosclerotic metabolites.
INTRODUCTION
Otosclerosis is a chronic pathological process leading to the formation of spongy bone and fixation of the stapes footplate in the oval window [1] . With an incidence of 1%-2% in the white population, otosclerosis is among the most common causes of acquired hearing impairment [2] .
In addition to progressive hearing loss, subjective tinnitus is one of the primary symptoms of the otosclerosis development [3][4][5][6] . The pathophysiology behind the tinnitus has not been clearly explained, despite ongoing scientific research. The "exchange hypothesis" of tinnitus formation in otosclerosis assumes that the newly formed bone tissue, because of its rich vascularization, leads to pulsatile tinnitus [7] . According to Ismi et al. [6] , tinnitus may be a consequence of the reduction of the inner ear fluid vibration or the production of toxic otosclerotic metabolites.
Preoperative measurement of the incidence of tinnitus in the adult population with otosclerosis is important because the number of patients treated for otosclerosis is increasing, suggesting that not only hearing loss but also tinnitus is a significant problem. In terms of treating otoslerosis, current approaches should not only aim to close the air-bone gap, but also to reduce or eliminate coexisting tinnitus [9,10] . To the authors' best knowledge, no comprehensive study of patients qualified for stapes surgery has been published yet, which, by using a battery of validated research tools, assessed the distress caused by tinnitus. The aim of this study is therefore to evaluate the prevalence and severity of preoperative tinnitus among a group of consecutive adult patients with otosclerosis, using standardized research tools.
MATERIALS AND METHODS
The study included patients qualified for treatment of otosclerosis by stapes surgery. The main inclusion criteria were age ≥18 years, suspected otosclerosis, and no previous stapes surgery (stapedotomy) in the ear eligible for surgery. We excluded patients whose intraoperative images excluded otosclerosis, or who needed a surgical procedure other than stapedotomy. Tinnitus was diagnosed as clinically relevant when it occurred at least once a week and lasted at least 5 minutes. According to the Clinical Practice Guideline on Tinnitus (American Academy of Otolaryngology-Head and Neck Surgery), tinnitus lasting longer than 6 months is classified as chronic [11] .
The research was conducted as a part of a standard diagnostic evaluation preceding the stapes surgery. The pure-tone audiometry was conducted in every participant of the study. The mean hearing thresholds for air conduction and bone conduction were determined at 500, 1000, 2000, and 4000 Hz. The air-bone gap was defined as the difference between the average bone conduction threshold and air conduction threshold.
Three validated questionnaires-The Tinnitus and Hearing Survey (THS-POL), Tinnitus Handicap Inventory (THI-POL), and Tinnitus Functional Index (TFI-Pl)-were used to assess the tinnitus complaint in patients reporting its occurrence. The acronyms and abbreviations are listed in Table 1.
The Tinnitus and Hearing Survey (THS) was published by Henry et al. [12] and adapted into Polish by Raj-Koziak et al. (as THS-POL) [13] . It is a quick and easy screening tool for differentiating hearing loss and tinnitus problems. The sum of the results obtained in Parts A and B allows the clinician to effectively diagnose the patient and choose a treatment direction.
Statistical Analysis
A statistical analysis was performed using The Statistical Package for the Social Sciences (SPSS) (IBM Corp.; Armonk, NY, USA) v. 24 program. Variables with a non-normal distribution were analyzed using non-parametric tests: the Kruskal-Wallis test and Spearman's correlation coefficient. The Mann-Whitney U test was used to compare two independent groups. The p<0.05 were considered statistically significant.
Prevalence and Severity of Tinnitus
The study group consisted of 157 patients: 106 women and 51 men. Seventy-one women and 36 men reported preoperative tinnitus, which was 68.2% of the study group. Tinnitus was equally common among women and men (67.0% of women and 70.6% of men reported chronic tinnitus). The average duration of tinnitus was 81.5 months (minimum 6, maximum 360). Fifty-six patients reported bilateral and 51 patients reported unilateral tinnitus (only in the ear qualified for surgery).
The THS-POL questionnaire indicated that 76.6% of patients had a greater problem with hearing loss than with tinnitus. For 13.1% of patients, tinnitus was more of a problem than hearing loss, and for 10.3%, the negative effects of hearing loss and tinnitus were on the same level.
Tinnitus and Gender
The average tinnitus severity scores from THI-POL and TFI-Pl are shown in Tables 2 and 3, respectively. The Mann-Whitney U test showed that there were no significant differences between women and men in tinnitus severity for subscales of THI-POL and TFI-Pl.
Tinnitus and Age
Participants were divided into five age groups at 10-year intervals. The average tinnitus severity scores for each age group are presented in Table 4. The correlation between age (years) and tinnitus severity was also investigated: the statistical analysis showed no relationship between age and severity of tinnitus for TFI-Pl (χ 2 = 3.72; p=0.445) or THI-POL (χ 2 =4.42; p=0.352). Only for the emotions subscale of the TFI-Pl questionnaire was a significant difference found: χ 2 =14.34; p=0.006.
Tinnitus, Gender, and Age
The results of tinnitus severity for women and men are presented in Tables 5 and 6, respectively. Based on both TFI-Pl and THI-POL, the severity of tinnitus increased with age in women, but not in men. For each gender, Spearman's correlation coefficient was used to determine the relationship between age and tinnitus severity. A significant correlation was found for women on the functional subscale of THI-POL (rho=0.28; p=0.020), meaning that as women get older, they experience greater tinnitus severity in the domain of functioning. The TFI-Pl questionnaire showed that older women were particularly susceptible to tinnitus severity in the following areas: hearing (rho=0.29; p=0.013), quality of life (rho=0.34; p<0.01), and emotions (rho=0.33; p<0.01). In men, the only relationship between age and severity of tinnitus was on the relaxation subscale of TFI-Pl (rho=−0.39; p=0.016). With advancing age, men perceive tinnitus as less troublesome in terms of opportunities for rest (relaxation).
In summary, although the total tinnitus severity result did not differ significantly between men and women (p>0.05), both TFI-Pl and THI-POL indicate that tinnitus severity increased with age in women, while in men, it decreased, but only in some areas.
Tinnitus and Audiometric Results
The relationship between audiometric results and tinnitus severity was analyzed. loss in 31 (29%). The average air conduction threshold was M=54.3 dB (SD=15.9), and the bone conduction threshold was M=25.5 dB (SD=12.6). The average air-bone gap was 28.8 dB (SD=8.7). There was no correlation between audiometric results and tinnitus severity measured by TFI-Pl and THI-POL (Tables 7 and 8, respectively). Only for the auditory subscale of the TFI-Pl questionnaire showed a weak relationship with air and bone conduction thresholds.
DISCUSSION
Preliminary results of our questionnaire study on 157 adults with otosclerosis have shown that 68.2% of patients experience chronic tinnitus before surgery. In the literature, the prevalence of tinnitus in otosclerosis has been determined to be 60%-90%. The prevalence of tinnitus in our study is similar to the results by Gristwood and Venables [19] and Bagger-Sjöback et al. [20] , who have used the largest research material.
In the Gristwood and Venables study of 1,014 patients, chronic tinnitus was preoperatively diagnosed in 65% of participants. Tinnitus was diagnosed as chronic when it lasted persistently longer than 3 months. This is the only scientific report focused exclusively on the prevalence of preoperative tinnitus in otosclerosis, although the authors did not assess tinnitus severity. The authors investigated the relationship between the results of pure-tone audiometry and the presence of tinnitus. Interestingly, the proportion of patients with tinnitus decreases with the increase in the mean level of air and bone conduction thresholds. The authors also did not explain why the proportion of tinnitus cases initially rises with the air-bone gap level but then falls again at the top level. In our study, there was a lack relationship between audiometric results and tinnitus severity. Although tinnitus often accompanies hearing impairment [21] , the correlation between tinnitus severity and hearing thresholds is not obvious. It can be explained by the fact that tinnitus perception and reactions to it are subjective phenomena. With respect to the general population, some authors have attempted to evaluate the potential link between tinnitus distress and an underlying hearing loss. Savastano [22] analyzed the clinical characteristics of tinnitus both in normal hearing subjects and in patients with hearing loss. The hearing impaired patients reported bigger tinnitus discomfort (measured by visual analogue scales, VAS); however, tinnitus handicap (measured by THI) was more severe in normal hearing subjects than in hearing impaired patients. Moon et al. [23] examined the role of hearing loss on the tinnitus symptoms severity in a group of 1,705 patients. Among the groups with low anxiety sensitivity, awareness, and loudness of tinnitus were significantly greater among patients with hearing loss than among those with normal hearing. A large cross-sectional study conducted in South Korea including 11, 266 people investigated the relationship between tinnitus, hearing loss, and the quality of life [24]. In this study, no significant association between the group with hearing loss without tinnitus and the quality of life was noted. The results obtained by Aazh et al. show a significant relationship between the pure-tone average (PTA) and the tinnitus loudness as measured by VAS [5] . However, the relationship was very weak (r=0.022; p<0.001), and statistical significance was probably achieved due to a large sample size. The authors conclude that tinnitus severity and the impact of tinnitus on life were more strongly correlated with tinnitus loudness than PTA. It is worth mentioning that Raj-Koziak et al. [25] demonstrated that the evaluation of tinnitus loudness with VAS could be a useful tool in diagnosing patients and measuring the effects of treatmentbut only in patients with normal hearing. The authors suggested that VAS measures of tinnitus loudness in patients with hearing loss might be over-reported because of overlapping complaints related to tinnitus and hearing loss. We can speculate that the assessment of tinnitus severity in otosclerosis patients can only be made by self-report questionnaires. In the study by Bagger-Sjöback et al. on 135 subjects eligible for stapes surgery, 68% reported tinnitus. Bast et al. [26] used 53 patient records to establish the presence of tinnitus in over 80% of their subjects. Similarly, Ismi et al. [6] found that in a group of 69 patients, 87.1% reported tinnitus. The highest percentage of tinnitus, some 90% of patients, was reported in studies by Sobrinho et al. [27] and Rajati et al. [5] , who studied 48 and 29 patients with otosclerosis, respectively.
Based on the results from the TFI-Pl and THI-POL questionnaires, we have shown that over 40% of adult otosclerosis patients eligible for surgery stapes experience tinnitus on a severity rated as a moderate to very large problem. Therefore, this group of patients expects surgery to not only improve their hearing, but also to reduce the annoyance associated with tinnitus.
Due to the use of slightly different methods and techniques to measure the severity of tinnitus in otosclerosis, a meta-analysis of our results and those from other scientific reports is not possible. Ayache et al. [28] used a self-created 4-point scale (slightly bothersome, bothersome, irritating, unbearable) to evaluate the severity of tinnitus. In another three publications, VAS was used to assess the distress caused by tinnitus [20,27,29] . Dewyer et al. [30] used VAS to assess the volume of tinnitus, which correlates with the severity of tinnitus as measured by the TFI questionnaire, although they did not provide actual data on the severity of preoperative tinnitus as assessed by the TFI. Bast et al. [26] in a study of tinnitus distress used the Tinnitus Questionnaire created by Goebel and Hiller [31] .
Subsequent researchers have adopted the same measurement tools as we used to evaluate the preoperative severity of tinnitus. Rajati at al. [5] used the THI questionnaire to measure the intrusiveness of tinnitus in a group of 26 patients. The authors classified their results according to the 5-point scale of Newman (1996) [14] : 23.1% of the patients fell into the first and second degree of tinnitus severity, 42.3% the third degree, 34.6% the fourth, and no patient reached the fifth degree. These results differ from ours and may be due to the small number of patients they used. Chang and Cheung [32] studied a group of 26 patients with otosclerosis and assessed the severity of their tinnitus using the TFI questionnaire. Unfortunately, no detailed analysis of preoperative tinnitus severity was reported, and although the work referred to the tinnitus severity classification proposed by Meikle et al. [17] , the results presented differed from the original source.
The available scientific literature on tinnitus severity in otosclerosis appears to be based largely on questionnaires dedicated to evaluating particular aspects of this ailment. However, in most of these studies, there is no information about whether the research tools used were validated. For the sake of reliability, it is desirable to cross-culturally adapt questionnaires in accordance with international validation guidelines. Numerous scientific reports on otosclerosis indicate that tinnitus more often affects women than men [4,5,19,[26][27][28]30] , and our research confirms this. The number of women included in our study was nearly twice that of men, but we found that subjective tinnitus was equally common in both genders. The literature on the association between gender and tinnitus in otosclerosis is inconclusive. Some authors claim there is no gender effect [5,27,28,33] . However, Deggoju et al. [29] found that women with otosclerosis are much more likely to have tinnitus than the general population and also than men having otosclerosis. Gristwood and Venables [19] showed a correlation between gender and the occurrence of preoperative tinnitus in otosclerosis: nearly 70% of 663 women and 56% of 351 men reported tinnitus before surgery. The authors did not present any hypotheses on why there was a higher prevalence of tinnitus among women.
In our study, the severity of tinnitus as measured by TFI-PI and THI-POL was not significantly different between men and women, although higher scores were observed in women. It is possible that this difference was due to nearly double the participation of women. Rajati et al. [5] also found no significant gender effect in preoperative tinnitus severity. The findings of Sobrinho et al. [27] were similar, although in the group of patients with the highest preoperative tinnitus severity (defined as 7-10 VAS points), 16 of 19 patients (84.2%) were women.
In terms of patient age, this factor also does not seem to be a predictor of the perceived severity of preoperative tinnitus. However, the TFI-Pl questionnaire indicated that the severity of tinnitus did increase with age on the TFI-Pl emotions subscale, which gauges the levels of feelings such as fear, anxiety, nervousness, and depressive states. The studies by Sobrinho et al. [27] and Rajati et al. [5] also showed no relationship between age and the severity of tinnitus.
We observed a clear correlation between age, gender, and the severity of tinnitus. In women, the severity of tinnitus increased with age, while it decreased in men. The THI-POL questionnaire showed that as women became older, tinnitus progressively impaired their functioning, meaning social, professional, and physical areas in which problems of disorientation, irritability, weariness, and increased stress arose. The TFI-Pl also pointed to the importance of the age factor for women in terms of its impact on the quality of life and emotions associated with tinnitus. We therefore hypothesize that older women are more likely to have problems with adapting to tinnitus, whereas in men, the ability to adapt to tinnitus increases with age.
Although providing valuable knowledge about tinnitus among patients with otosclerosis, the study has several limitations. One of them is assessing the tinnitus complaint only in a group of patients qualified for stapes surgery, which may not reflect the tinnitus complaint met in patients with smaller air-bone gaps (at the initial stage of otosclerosis development) or those not willing to undergo the surgical procedure to improve hearing. Additionally, our current findings concern only the preoperative period, without providing information on the effectiveness of stapes surgery in the postoperative tinnitus cessation. However, knowing that tinnitus is such a prevalent and significant complaint, such investigation is planned as a next step of our research.
CONCLUSION
A major lack of information on the prevalence and severity of tinnitus in otosclerosis has prompted us to conduct, to the best of our knowledge, the first prospective study on the subject. The main con-sequence of otosclerosis is progressive hearing loss, which also leads to tinnitus, and they both significantly reduce the quality of life. It is therefore advisable to continue studies in this area, since knowledge about tinnitus in otosclerosis can be very useful in qualifying patients for stapes surgery and meeting their expectations about outcomes. This work is a first step toward the next stage of research, which is to evaluate changes in the severity of tinnitus after surgical treatment for otosclerosis.
Ethics Committee Approval: The study was designed and conducted according to the Declaration of Helsinki, with the study protocol reviewed and approved by the Institutional Review Board of Institute of Physiology and Pathology of Hearing (IFPS/KB.05.2017).
Informed Consent: Informed consent was obtained from all individual participants included in the study. | 2019-08-17T13:04:17.598Z | 2019-08-01T00:00:00.000 | {
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225304461 | pes2o/s2orc | v3-fos-license | CONVERSION OF LINOLEIC ACID TO DIFFERENT FATTY ACID METABOLITES BY Lactobacillus Plantarum 13-3 AND IN SILICO CHARACTERIZATION OF THE PROMINENT REACTIONS
Lactobacillus plantarum strains have been extensively used in food processing and preservation. L. plantarum also has the potential to convert polyunsaturated fatty acids, e.g. linoleic acid (LA) into bioactive and less toxic fatty acid metabolites. The objective of this study was to assess the capability of probiotic L. plantarum 13-3 to convert Linoleic Acid (LA) to different fatty acid metabolites in the medium supplemented with differential concentrations of LA, and the relevant reactions were characterized by in silico calculation. L. plantarum 13-3 was grown in MRS medium containing LA from 1% to 10%, and the fatty acid metabolites formed in the medium were identified and quantitated by GC-MS and in silico studies were done to confirm the enzymatic reactions involved in its conversion. The results showed that L. plantarum 13-3 could convert LA at different concentrations to 5 different fatty acid metabolites i.e, (Z)-Ethyl heptadec-9-enoate, 9,12-Octadecadienoic acid (Z, Z), methyl ester, Octadec-9-enoic acid, cis-11,14-Eicosadienoic acid, methyl ester and (Z)-18-Octadec-9-enolide. Among these metabolites, the formation of an long chain fatty acid Octadec-9-enoic Acid, also known as 18:1, N-9 or Delta(9)-Octadecenoic acid, is classified as a member of the Long-chain fatty acids in media supplemented with 4% to 10% LA, is being reported for the first time. Putative candidate enzymes involved in biotransformation of LA into fatty acid metabolites were identified in whole genome of L. plantarum 13-3, sequenced previously. In silico studies confirmed that several enzymes including the linoleate isomerase, acetoacetate decarboxylase and oxidoreductase may be involved in biotransformation of LA into fatty acid metabolites. These enzymes could effectively bind the LA molecule mainly by forming hydrogen bonding between the acidic groups of LA and the proline residues at the active sites of the enzymes validating the putative reacting partners.
INTRODUCTION
Linoleic acid (c18:2 n-6) (LA) the most abundant polyunsaturated fatty acid (PUFA), also known as an essential fatty acid and it is a significant constituent of low-density lipoproteins [1]. LA cannot be produced endogenously in animals and hence its only source is via dietary intake [2]. Algae and plants contain adequate amounts of the Δ12and Δ15-desaturase enzymes and as a result LA and α-linolenic acid are the most widespread fatty acid found in plant tissues and oils.
As a PUFA, LA can be oxidized through endogenous enzymes and reactive oxygen species in the circulation. Metabolites of LA are formed as a result of oxidation by the action of endogenous enzymes. LA and its byproducts are known to exhibit various biological effects and they are involved in metabolic disorders and cancer [3][4][5][6][7]. The irony of an essential fatty acid contributing in both beneficial and pathological processes may be explained by its conversion to biologically active metabolites [8][9]. Several metabolic processes in the host are regulated by gut microbiota, some of these processes include lipid metabolism, glucose metabolism and homeostasis [10]. The gut microbiota is also capable of performing various processes that cannot be carried out by the host and these processes can give rise to microbially produced or modulated metabolites which works as metabolic substrates and signaling molecules in the host, with key implications for host metabolism and health [11]. Many bacteria have been noticed that their growth has been inhibited by Polyunsaturated fatty acids (PUFA) such as Linoleic acid (LA; 18:2Δ9Z,12Z). PUFA in high concentration is toxic and may block native fatty acid biosynthesis by the inhibition of enoyl-ACP reductase [12][13][14][15]. Bacteria have developed a detoxification mechanism and are capable of enzymatically hydrogenate PUFA by completely reducing the double bonds on the carbon chain. i.e., bio hydrogenation, producing non-toxic saturated fatty acids as the end product [16]. This process includes various steps and has been greatly described for LA and oleic acid (OA; 18:1 Δ9Z) that are converted to non-toxic saturated stearic acid (SA; 18:0) by rumen microbiota [16][17][18][19].
Lactic acid bacteria (LAB) are the most important and common starter cultures used in fermented dairy products, and they may originate from the microflora of raw milks (e.g., bovine, ovine, caprine) but most commonly are inoculated intentionally during product manufacture.
LAB are generally regarded as safe (GRAS) microorganism which have been traditionally used in food fermentation for a long history. Traditionally fermented dairy products are considered as a key source of functional microorganism, e.g. LAB and ingredients [20]. Many LAB strains are able to produce various bacteriocins, exopolysaccharides, fatty acids, etc. engaging their useful health effects. Many LAB strains have shown different promising bioactivities on human health, including antimicrobial activity, prevention and treatment of diarrhea, relief of symptoms caused by lactose intolerance, anti-mutagenic and anti-carcinogenic activities and stimulation of the immune system [21]. However, the uncertainties of the influence from these LAB strains on the quality of functional foods and their bioactivity keeping in food matrix frequently prove their application in modern food industry [22].
L. plantarum is homofermentative having the competency to transform growth-inhibiting free polyunsaturated fatty acids, e.g. linoleic acid (LA) at a comparatively higher concentration, into bioactive conjugated LA (CLA) and other less toxic fatty acids metabolites e.g. hydroxy fatty acids, oxo fatty acids, conjugated fatty acids and saturated trans fatty acids, as the intermediates to eventually produce saturated monoenes (OA and trans-vaccenic acid) [34][35][36].
In silico analysis of the whole genome sequences of L. plantarum 13-3, exposed the presence of many enzymes including acetoacetate decarboxylase, Oxidoreductase and linoleate isomerase.
We validated our findings by employing in silico analysis to characterize the relevant reactions involving the enzymes that were identified using the whole genome sequence of L. plantarum 13-3. Our findings would have great prospect for human nutrition, nutraceuticals and food industry and will pave way into identifying the relevant reactions and doses for biotransformation of LA into desirable fatty acid metabolites.
Microorganism and growth conditions
L. plantarum 13-3 isolated from Tibetan Kefir preserved in the culture bank of Dairy Laboratory in Beijing Technology and Business University of China was repeatedly revived for three times at 37 o C in MRS medium (Beijing Aoboxing Co Ltd) containing 2.0% glucose, 1.0% meat extract, 1.0% tryptone, 0.5% yeast extract, 0.1% Tween 80, 0.2% K2HPO4, 0.5% sodium acetate, 0.2% diammonium citrate, 0.02%, MgSO4 x 7H2O and 0.005%, MnSO4 x H2O was used in this study. Distilled water was used as solvent for dissolving medium components and medium was adjusted to pH 5.5 and sterilized at 121 o C for 15 minutes. The fresh MRS medium added with different concentrations of LA was inoculated with 1% of the activated culture of L. plantarum 13-3 for growth and production of metabolites at 37 o C.
Spectrophotometric determination of fatty acid metabolites
The culture sample were centrifuged at the speed of 13,000 ×g for 5 min at 4 o C and 1 mL of the supernatant was mixed with 2 mL of isopropanol. After addition of 1.5 mL of hexane, the mixture was extensively vortexed in order to extract the lipids and then allowed to stand for 5 min. The hexane layer was collected and the absorbance was measured at 233 nm. The fatty acid metabolites were extracted by using hexane/isopropanol (2:1, v/v) solution at room temperature, and the extracts were washed with distilled water and then dehydrated with anhydrous sodium sulfate [37].
Extraction of fatty acids from the medium.
For the analysis by gas chromatography (GC), the culture samples were centrifuged at 1900 rpm for 5 minutes at 4 o C to remove the cells. An internal standard (C17:0, heptadecanoic acid, 98% pure; Macklin) was added to 5 ml of the supernatant fluid to give a final concentration of 0.15 g/ml. Then 5 mL of isopropanol was added and vortexed for 30 s. Subsequently 2 mL of isopropanol was added and vortexed for 30 s. Lastly, 5 mL of n-hexane was added to this mixture, vortexed for 3 min, incubated for 30 min, and centrifuged at 1900 rpm for 5 min. The upper hexane layer containing fatty acid methyl esters (FAME) was collected and was dried under a steam of liquid nitrogen [38].
Gas chromatography and mass spectrometry (GC-MS)
The GC-MS analysis was done by using Shimadzu GC-2010 instrument coupled with a Dual Stage TMP (Ultra) mass spectrometer. The FAME sample of 2 μL was injected in a split mode, set at 10:1 split ratio at 250℃. The carrier gas was helium at a constant flow rate of 1mL/min. The separation was conducted on a highly polar (TR-Wax MS, 30 m length × 0.25 mm i.d. × 0.25μm thickness) and fused silica capillary column (Thermo Fisher Scientific). The initial oven temperature was held at 170℃ for 1 min, then increased at 0.8 ℃/min to 200℃. The temperature of line transfer was at 250°C, and the ion source was controlled at 200°C. The MS detector was operated in an electron ionization (EI) voltage of 70 eV under a mass scan range of 33-450 amu (m/z).
Identification of fatty acid metabolites
Chemical identification was conducted by comparison of the mass spectra (MS) of the peaks with those found in the National Institute of Standard and Technology library (NIST, 2014).
In silico characterization of conversion of LA to various fatty acid metabolites by L. plantarum 13-3
Enzymes responsible for catalyzing the relevant reactions for conversion of LA to different fatty acid metabolites in L. Plantarum 13-3 were identified by analysis of the whole genome sequence (GCA_004028315.1) sequenced earlier using SWISS-MODEL that is a fully automated protein structure homology modelling server [39] The protein module was visualized by protein visualizing program Discovery Studio 3.5 [40]. The LA molecule was drawn in ChemSketch v15.0.9 (Chemaxon) assigned with proper 2D orientation, and the structure of each compound was analyzed for connection error in bond order. Energy of the molecules was minimized using Avogadro [41] with MMFF94 force field. Docking studies calculations were performed by Autodock Tool [42]. Protein (LAI, DH and DC) and ligands (LA) structures were converted to pdbqt file by MGL Tools 1.5.6 rc 3 . The interactions of complex protein-ligand conformations were analyzed using Autodock Tools 4.2 [42]. and Discovery Studio 4.1 [40].
Statistical Analysis.
Statistical analysis of the data was done using ANOVA SAS version 9. Evaluation of the significance of differences between groups was performed with one-way ANOVA as mentioned in figure legends.
RESULT AND DISCUSSION
L. plantarum 13-3 exhibited similar growth pattern at the concentrations of LA from 1% (w/v) to 10% (w/v) as shown in Fig. 1A. For all the concentrations of LA, it was observed that L. plantarum 13-3 increased its growth up to 24 h, subsequently entered stationery phase till 36 h, and then the growth decreased till 48 h. There was slightly increase in the growth of the strain with the increase of the LA concentration. On the contrary, many other LAB strains were reported to be inhibited to different extent by LA and their tolerance to LA varied [37,[39][40]. While earlier studies showed that even lower LA levels (25 µg/ml) could inhibit bacterial growth [37], L. plantarum 13-3 was able to grow well at the concentration of LA up to 10 % (w/v), indicating its relatively high tolerance to LA. (Fig1B). showed that production of total fatty acid metabolites gradually increased with the increase of LA concentration from 1% (w/v) to 10% (w/v) as indicated by the increased absorption at the wavelength of 233 nm. This indicated that L. plantarum 13-3 exhibited high tolerance to LA by converting LA to less toxic fatty acid metabolites. Previous researcher also advocated that conversion of free LA to LA analogues might function as a detoxification mechanism in bacteria and a stronger LA tolerance indicated a higher productivity of LA analogues/metabolites [37,39,[45][46]. Furthermore, more fatty acid metabolites were produced during the stationery phase of growth of L. plantarum 13-3 and the production reduced when the death phase started. Similar findings were also reported with other microbial producers of LA fatty acid metabolites/analogues [47][48]. Significantly, it should be highlighted that the spectrophotometric method does not differentiate between isomers of CLA since it is based on measurement of the conjugated double bond in the fatty acid [37].
Identification of fatty acid metabolites by GC-MS
The fatty acid metabolites produced by L. plantarum 13-3 at different concentrations of LA were identified and quantitated by GC-MS ( Table 1). Total of 5 fatty acid metabolites were identified including one long chain fatty acid, octadec-9-enoic acid C18H34O2, one ethyl ester, (Z)-ethyl heptadec-9-enoate C19H36O2, two methyl esters 9,12-octadecadienoic acid (Z,Z)-methyl ester C19H34O2 also known as methyl linoleate which is found in clove and is mainly used as a flavoring agent and cis-11,14-eicosadienoic acid, methyl ester, and (Z)-18-octadec-9-enolide as shown in Table 1. The following compounds, (Z)-ethyl heptadec-9-enoate, 9,12-octadecadienoic acid (Z, Z)-methyl ester, and cis-11,14-eicosadienoic acid methyl ester were identified under the all concentration of added LA as shown in Table 1. These findings proved that the ability of L. plantarum 13-3 to produce the above mentioned fatty acids. While, the octadec-9-enoic acid was produced by L. plantarum 13-3 under the concentration of added LA ranged from 4 % to 10 %. Finally, (Z)-18-octadec-9-enolide was produced only at 10 % of added LA as listed in Table 1.
Name
RT 1% 2% 3% 4% 5% 6% 7% 8% 9% 10% (Z)-Ethyl heptadec-9-enoate 15.112 + + + + + + + + + + Linoleate isomerase is the enzyme which has the responsibility for the conversion of one isomeric form of ligand to another form via enzymatic mechanism. Isomers are different form of the same ligands having same molecular formula but different structural formula having different 3D orientation in space. The genome of L. plantarum 13-3 was blasted to discourse different linoleate isomerase giving many hit, and best hit was selected for further study.
The Conversion of linoleic acid (LA) into several metabolites were studied by using computational approach. LA was optimized by using Avogadro and saved into PDB format. Linoleate isomerase gene was found in L. plantarum 13-3 which was blasted by using NCBI platform and then module by using Swiss model. Linoleate isomerase is a monomer protein having tertiary structure which consists of its backbone, side chain, hydrophobic moiety, hydrophilic moiety, acidic group and basic group shown in figure 2. The collective interaction of the linoleate isomerase with LA was carried by using Auto-dock Vina Program. The optimized structure of linoleate isomerase and linoleic acid was used for Autodock study. After docking it was observed that Carboxylic Acidic moiety of LA formed Hydrogen bonding with Lysine (LYS A:549) and also showed electrostatic and Vander wall interactions. As Linoleate isomerase is NADPH dependent enzyme so Hydrogen from NADPH attack on the double bond of the LA ultimately the reaction of LA with Linoleate Isomerase and NADPH leading to the formation of different metabolites.
Acetoacetate Decarboxylase
Acetoacetate decarboxylase belongs to the class of enzymes which plays a vital role in solvent production by catalyzing the decarboxylation of acetoacetate moieties, producing acetone and carbon dioxide. The production of solvent acetone by enzyme acetoacetate decarboxylase containing bacteria was utilized in large-scale industrial syntheses in the first half of the twentieth century. Recently we have explored in this study that beyond it, it also plays important role in fatty acid chemistry by converting LA into different fatty acid metabolites.
The genome of L. plantarum 13-3 was blasted for the search of different Acetoacetate Decarboxylase giving many hit, and best hit was selected for further study. The Protein Structure of Acetoacetate Decarboxylase of L. plantarum 13-3 module by Swiss model is shown in figure below. The structure of enzyme Acetoacetate Decarboxylase having ligand in the active site is given below in figure 5. The Acidic group of LA formed two hydrogen bonds with Lysine 114 (LYS: A114) and the other with Arginine 106 (Agr: A106). The acidic moiety holds by enzyme by hydrogen and electrostatic interaction, which provide medium for the double bond transfer reaction.
Oxidoreductase
Oxidoreductase is a class of enzyme that are involved in catalyzing the reaction which involve the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. So these enzymes usually involve the transfer of charge from catalyzing molecule leading to oxidation or reduction. This group of enzymes usually NADP or NAD+ dependent enzyme which use it as cofactors. Dehydrogenase is tetramer, composed of monomer A, B, C and D. The active site residue and substrate in it is reported in figure 7 given below.
CONCLUSION
L. plantarum 13-3 was shown to be a competent candidate for converting LA from 1% to 10% (w/v) to various fatty acid metabolites such as Z)-Ethyl heptadec-9-enoate, 9,12-Octadecadienoic acid (Z,Z) , methyl ester, Octadec-9enoic acid, cis-11,14-Eicosadienoic acid, methyl ester and (Z)-18-Octadec-9enolide. Main reactions involved in this conversion include isomerization, dehydrogenation and reduction as confirmed by the in silico analysis. The putative linoleate isomerase and dehydrogenase catalyzing the relevant reactions were identified using the whole genome sequence of L. plantarum 13-3. These two enzymes were shown to bind the LA molecule at their active sites mainly by formation of hydrogen bonding between the acidic group of LA and the proline residues of the enzymes. Further study is required to investigate the role of the conversion from LA to its various fatty acid metabolites by L. plantarum 13-3 physiologically and the implications for exploring the potentiality in functional foods. | 2020-10-28T19:09:26.405Z | 2020-09-10T00:00:00.000 | {
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4561913 | pes2o/s2orc | v3-fos-license | Downregulation of SNAIL sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis by regulating the NF-κ B pathway
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the second most lethal cancer worldwide. Evidence has shown HCC cell resistance to TRAIL-mediated apoptosis. In a previous study, we verified that silencing SNAIL downregulated the growth of HCC cells. In addition, the mechanism of resistance to TRAIL in HCC cells was connected with the activation of nuclear factor-κB (NF-κB). Thus, it was hypothesized that the downregultaion of SNAIL sensitizes HCC cells to TRAIL-induced apoptosis by regulating the NF-κB pathway. In the present study, the most effective lentiviral vectors carrying shRNA against SNAIL were selected and adenoviral vectors harboring TRAIL were constructed. The expression of SNAIL and TRAIL was detected by quantitative PCR and western blotting. HCC cell viability and apoptosis were assessed using an MTT assay and the Hoechst test. To determine how to sensitize HCC cells to TRAIL-induced apoptosis after silencing SNAIL, p53 was assessed by western blot analysis. We also investigated the expression of Bcl-xL, cIAP2, survivin and Raf-1 protein using western blot analysis and the apoptotic degree of HuH-7 cells was detected using the Hoechst test following the suppression of each gene, which was a possible molecular mechanism to sensitive TRAIL-induced apoptosis through the downregulation of SNAIL in HCC cells. Silencing SNAIL resulted in increased apoptosis by enhancing sensitization to TRAIL in all the HCC cells. Additionally, p53 protein was upregulated in HuH-7 cells. Expression of Bcl-xL, cIAP2, survivin and Raf-1 was downregulated following silencing of SNAIL, while down regulation of any of the proteins contributed to SNAIL suppression enhancing HCC cell sensitivity to TRAIL-induced apoptosis, with the exception of cIAP2. The results demonstrated that silencing SNAIL can sensitize TRAIL-induced apoptosis in HCC cells by upregulating p53 protein and by regulating related genes of the NF-κB pathway such as Bcl-xL, survivin and Raf-1.
Introduction
Hepatocellular carcinoma (HCC), as the most common type of primary liver cancer, is the sixth most common cancer globally, with a prevalence of ~600,000 individuals worldwide.HCC is the second most lethal cancer worldwide and one of the most rapidly growing cancer types in Asia, particularly in China (1,2).Reaction of biochemistry and gene control of apoptosis are important in hepato carcinogenesis.Failure of apoptosis further promotes the development of cancer, owing to tumor occurrence.The majority of HCC cells are markedly resistant to the stimuli of inducing apoptosis, which can lead to HCC cell anti-apoptosis (3).Many factors prevent HCC cells from apoptosis, including anti-apoptotic members Bcl-2/xL (4), survivin and cyclooxygenase (COX)-2 (5,6).The abnormal expression of these factors contributes to resistance to HCC cell apoptosis, resulting in loss of tumor control and patient death.Therefore, identification of a new mechanism to convert resistance to apoptosis and promote apoptosis for the treatment of HCC is crucial.
Downregulation of SNAIL sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis by regulating the NF-κB pathway
with activation of the NF-κB pathway, upregulation of apoptotic inhibitors, such as cFLIP, IAP and anti-apoptotic molecules, such as certain Bcl-family members (12)(13)(14)(15).TRAIL is involved in inhibition of cancer.Thus, the upregulation of HCC cell sensitization in TRAIL-induced apoptosis by mediating the above targets is significant in HCC treatment.SNAIL, as a transcriptional repressor, has a structure of zinc finger (ZF) which plays an important role in physiological processes such as embryonic development, and in a wide variety of pathologic processes (16,17).SNAIL has been found to enhance cancer invasion and metastasis in various malignancies (16,18,19).Similarly, SNAIL is crucial to the pathological progression of HCC.Evidence suggests that SNAIL contributed to tumor progression by inducing EMT to transform epithelial cells into mesenchymal ones (18).In addition, a study showed that SNAIL was induced and accelerated cell activity by repressing E-cadherin expression and upregulating MMP expression in HCC both in vitro or in vivo (19).Findings of another study showed that knockdown of SNAIL reduced proliferation and viability of HCC cells by increasing the expression of E-cadherin (20).All the aforementioned mechanisms suggested that SNAIL affects the viability and migration of HCC cells.Nevertheless, whether SNAIL affects the apoptosis of HCC cells remains to be determined.Thus, it is imperative to examine the characteristics of SNAIL with regard to the occurrence and progression of HCC, particularly in cell apoptosis.
RNA interference (RNAi) has emerged as a new and an indispensable tool for loss of gene function in eukaryotes in order for small-interfering RNA (siRNA), a class of synthetic short double-stranded RNA, to emerges at the appopriate time point (21,22).siRNA functions by inducing the silencing of gene by guiding endonucleolytic cleavage of message RNA (mRNA) or repressing its translation (23,24), while siRNA cannot maintain long-term silencing of the interfering gene.Thus, to induce the gene to stable and reversible silencing for a long period of time, short hairpin RNA (shRNA) was generated as a tool of the RNAi technique (21).A variety of viral and non-viral vectors can carry and express shRNA (22).Recently, studies verified successful construction of the lentivirus-shSNAIL vector, which could be infected into the HepG2 cell line to silence SNAIL (20).Adenoviral vectors harboring TRAIL were constructed by our laboratory previously (25), which all provide powerful support for our study.
In the present study, by silencing SNAIL via the utilization of RNAi in HCC cells, we investigated the effect of SNAIL deficiency for HCC cell apoptosis and the sensitization effect for TRAIL-induced apoptosis by downregulating SNAIL in HCC cells.The possible mechanism involved was also assessed.
Materials and methods
Cell line and cell culture.The human HCC HepG2, HuH-7 and HEK293 (human embryonic kidney cells) cell lines were purchased from the American Type Culture Collection (Manassas, VA, uSA).SMMC7721 was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.The cells were maintained in a humidified condition that contained 5% CO 2 at 37˚C and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine, followed by the addition of 100 u/ml penicillin and 100 µg/ml strepto mycin.
Construction of lentiviral and adenoviral vectors.The most effective lentiviral vectors carrying shRNA were previously screened in our laboratory (20).Adenoviral vectors harboring TRAIL and lentiviral vectors carrying shBcl-xL, shcIAP2, shSurvivin and shRaf-1 were previously constructed by our laboratory (20,25).Construction and purification of the vectors were performed as per the standard protocol.The titration of recombinant lentiviruses and adenoviruses was performed using a TCID50 assay on HEK293 cells.Recombinant lentiviral and adenoviral vectors were short for LV and Ad5, respectively.The cells were subsequently divided into the Mock, LV-shSNAIL, Ad5.TRAIL and Ad5.TRAIL + LV-shSNAIL groups.
Quantification by real-time PCR.Infected cells were collected and washed with phosphate-buffered saline (PBS).Total RNA was extracted from the cells of the target cells, and cDNA was generated with a PrimeScript ® RT reagent kit (Takara, Chiga, japan) at a total volume of 20 µl according to the manufacturer's instructions.cDNA was then used in each amplification reaction.Reactions were performed using SYBR ® Premix Ex Taq™ (Takara), under the following PCR conditions: denaturation at 95˚C for 30 sec, followed by 40 cycles of annealing at 95˚C for 5 sec and extension at 60˚C for 30 sec.Each sample was also subjected to melting curve analysis to confirm amplifica tion specificity.GAPDH was used as a control housekeeping gene.The expression of SNAIL was assessed by normalization of the cycle threshold (Ct) of these genes to that of GAPDH.A Ct value was obtained from each amplification curve by using the software provided by the manufacturer (Roche, Mannheim, Germany).
Cell proliferation detection.The analysis of HCC cell viability was determined by an MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay.HCC cells were plated in 96-well microtiter plates equally according to the density standards of 5x10 3 cells/well.Each well was provided suitable culture medium with the condition in 5% CO 2 at 37˚C until grown to a high density.Each group of cells was incubated for 24-96 h following treatment with various viruses.Subsequently, 5 mg/ml MTT was added to each well and incubated for 4 h under the same condition.The original culture of each well was abandoned.The crystal substances produced from the cells were dissolved in 150 µl dimethyl sulfoxide (DMSO) and agitated for 10 min.Cell viability was assessed as optical density (OD) at a wavelength of 490 nm for immune monitoring by the enzyme-linked immunosorbent spot assay (Bio-Rad, Hercules, CA, uSA).
Western blot analysis.Whole-cell lysates were obtained following centrifugation at 120,000 x g for 10 min of the target cells of all the groups.Total proteins were separated by electrophoresis on 12% polyacrylamide and transferred to 0.45 µm nitrocellulose (NC) membrane.The samples were washed three times using PBS after Ponceau S Staining kit dyeing.Bands were incubated with primary antibodies, rabbit polyclonal anti-SNAIL Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, uSA) and anti-TRAIL (Cell Signaling Technology, Danvers, MA, uSA), respectively.Conjugates were combined with secondary goat anti-rabbit alkaline phosphatase antibody (Abcam, Cambridge, MA, uSA), which was marked with HRP (horseradish peroxidase).GAPDH detection was performed using rabbit polyclonal anti-GAPDH and mouse monoclonal anti-GAPDH (Abcam).The fluorescent compounds were detected by ECL (Pierce, Rockford, IL, uSA).At the same time, primary antibodies, rabbit polyclonal anti-p53 (Cell Signaling Technology), anti-Bcl-xL (Cell Signaling Technology), anti-cIAP2 (Abcam, Cambridge, uK), anti-survivin (Cell Signaling Technology), anti-Raf-1 (Cell Signaling Technology), respectively, were applied to the membranes and the process was repeated as above.
Hoechst test.HCC cell lines were placed into 6-well plates and incubated with recombinant lentiviruses or adenoviruses, respectively.After 48 h, when the cells were infected, the medium was washed with PBS twice.The cells were then stained with Hoechst 33258 (25 µg/ml).Hoechst 33258 was excited by 405-nm violet diode.The percentage of apoptotic cells was analyzed using a fluorescence microscope.
Statistical analysis.The statistical analyses were performed using the Student's t test and one-way ANOVA to determine the significance.Results were presented as mean ± standard deviation (SD).P<0.05 was considered statistically different.
Expression of SNAIL in HCC cells.
In our previous study (20), we detected the expression of SNAIL in HepG2.Based on the results, LV-shSNAIL was found to suppress the expression of SNAIL at the mRNA and protein level in HepG2 cells (20).The mRNA level was measured by employing quantitative PCR, while western blot analysis was used to determine the protein expression level.The results verified that the level of SNAIL was obviously reduced following LV-shSNAIL infection in other HCC cells (Fig. 1), which was consistent with the anterior study (20).The findings showed that the construction of LV-shSNAIL was successful in silencing SNAIL expression in various types of HCC cells.
Cell viability and apoptosis analysis in HCC cells.Recent findings have shown that, suppressing SNAIL may inhibit invasion and metastasis in human HCC (26).Moreover, as described above, silencing SNAIL reduced viability in HCC cells.It is well known that the main function of TRAIL is the induction of tumor apoptosis, as identified by findings from our laboratory (25,27).Therefore, we first evaluated the status of TRAIL expression in different groups of three types of HCC cells, and the level of TRAIL was detected among the cells using western blot analysis.Analysis of the results showed that TRAIL expression was markedly increased following the addition of Ad5.TRAIL + LV-shSNAIL (Fig. 2).These data showed that TRAIL was effectively expressed following Ad5.TRAIL vector infection of HCC cells, and that knockdown of SNAIL increased TRAIL expression.
We also examined the effect of SNAIL and TRAIL on HCC by assessing the state of cell proliferation.In order to study the change in HCC cell viability for response to the SNAIL silencing and TRAIL, respectively, we detected cell viability in the four groups, from the point when reagents were added until 96 h, once every 24 h by MTT assay (Fig. 3).The extent of inducing apoptosis was also investigated.The samples were stained with Hoechst 33258 for 48 h and then analyzed by fluorescence microscopy (Fig. 4).The results revealed that silencing SNAIL or upregulated TRAIL decreased proliferation and increased apoptosis in HCC cells.
Inhibition of SNAIL enhanced TRAIL-induced apoptosis.
Since TRAIL-induced apoptosis was weakened in HCC cells as compared to other TRAIL-sensitive cells, previous studies focused on apoptosis induction by acting on TRAIL for HCC cells (28).Therefore, we examined the change of TRAIL-induced apoptosis in different groups.As shown in Fig. 4, HCC cell apoptosis was highest in the group infected with LV-shSNAIL and Ad5.TRAIL compared to the other groups.This result suggested that silencing SNAIL enhanced sensitivity that TRAIL induced HCC cell apoptosis.
Knockdown of SNAIL increased TRAIL-induced apoptosis by upregulating p53 expression.
Previous authors reported that p53 sensitized TRAIL-induced apoptosis in various types of cancer (29,30) and blocking SNAIL activated p53, which regulated apoptosis (31).To determine whether p53 was associated with silencing of SNAIL to increase TRAIL-induced apoptosis in HCC, we detected the expression level of p53 in different groups in HuH-7 cells.The results revealed that p53 expression was highest in groups to which LV-shSNAIL was added than those where LV-shSNAIL was not added (Fig. 5).
SNAIL silencing enhanced TRAIL-induced apoptosis by affecting the NF-κB pathway.
Resistance of HCC cells to TRAIL-induced apoptosis has been shown to be associated with Bcl-xL, cIAP2 and survivin factors, which are located in the downstream gene of the NF-κB pathway (14,15,32).Moreover, Hall et al suggested that blocking the activation of Raf kinase inhibited NF-κB binding to the Mcl-1 promoter thereby enhancing the sensitization of TRAIL-mediated apoptosis in cancer cells (13).Among them, Raf-1 kinase, a member of the Raf kinase family, is an important signaling molecule acting on the downstream kinases MEK and ERK, which pathway converges on NF-κB activation (13,33).Accordingly, we investigated the expression levels of these proteins in HCC cells to analyze the mechanism associated with the enhancement of TRAIL-induced apoptosis by SNAIL silencing.The results showed that Bcl-xL, cIAP2, survivin and Raf-1 expression were reduced following the addition of LV-shSNAIL than without its addition to HuH-7 cells, and particularly low in LV-shSNAIL + Ad5.TRAIL (Fig. 5).
In order to determine whether Bcl-xL, cIAP2, survivin and Raf-1 participated in the suppression of SNAIL to mediate HCC cell sensitivity to TRAIL-induced apoptosis, we used lentiviral vectors carrying shRNA to infect HuH-7 for the knockdown of Bcl-xL, cIAP2, survivin and Raf-1.The effect of TRAIL-induced apoptosis of HuH-7 cells was analyzed by fluorescence microscopy following the knockdown of each gene.Infection of LV-shBcl-xL, LV-shSurvivin or LV-shRaf-1 HuH-7 cells showed obvious sensitivity to TRAIL-induced apoptosis, whereas the sensitivity of shcIAP2 was not affected (Fig. 6).
In conclusion, Bcl-xL, survivin and Raf-1 downregulation was conducive to enhancement of HCC cell sensitivity to TRAIL-induced apoptosis by silencing SNAIL.In other words, silencing of SNAIL enhanced TRAIL-induced apoptosis by affecting the NF-κB pathway.
Discussion
In the present study, shSNAIL carrying lentiviral and adenoviral vectors harboring TRAIL induced SNAIL silencing and enhanced TRAIL expression.The results show that HCC cell apoptosis was increased whether via silencing SNAIL or enhancing the level of TRAIL, as confirmed by the cell viability results.The results also show that the sensitivity of TRAIL-induced apoptosis was increased when SNAIL was silenced.To examine the mechanism involved, we found that p53 performed a crucial role in the sensitization of HCC cells to TRAIL by downregulating SNAIL.Other mechanisms identified showed that silencing SNAIL enhanced TRAIL-induced apoptosis by affecting the NF-κB pathway.Therefore, enhancing the sensitization of HCC cells to TRAIL is essential for the promotion of HCC cell apoptosis.Moreover, SNAIL is a potential target that may overcome resistance of HCC cells to TRAIL-induced apoptosis, rendering it an improved therapeutic strategy for HCC.
As previously mentioned, most HCC cells were resistant to apoptosis in many stimuli, which included TRAIL.However, findings of recent studies (3,12,13,30) have shown that many targeted drugs alter their characteristics by acting on special targets.In the present study, we found that silencing SNAIL can increase the expression of p53, which alters the sensitization of HCC cells to TRAIL.On the other hand, it has been reported that p53 upregulated TRAIL death receptors (34,35).Our results have shown that silencing SNAIL upregulated p53.However, whether TRAIL death receptors are correlated with p53 remains to be determined.In addition, another study has shown that adenoviral-mediated transfer of p53 gene enhanced TRAIL-induced apoptosis in human HCC cells, not because of the induction of TRAIL death receptors, but due to the downregulation of cFLIP or XIAP (36), which resisted sensitization of some cell types to TRAIL-induced apoptosis (32,37).Thus p53 is an important gene that enhances the sensitization of HCC cells to TRAIL by downregulating cFLIP or XIAP under certain stimulation.Irrespective of the mechanism involved, p53 as a target is an important breakthrough point of HCC treatment.
Bcl-xL as an anti-apoptosis-associated protein member of the Bcl-2 family may play a very vital role in regulating the apoptosis of HCC (38).Overexpression of Bcl-xL contributes to TRAIL resistance in cancer, including HCC (14,(38)(39)(40).Given that Bcl-xL may be a bridge between SNAIL and TRAIL-mediated apoptosis in HCC, we further studied the relationship among them.The result showed that the expression of Bcl-xL was reduced by silencing SNAIL in HCC, and according to our experimental data, suppression of Bcl-xL by SNAIL silencing was able to increase the sensitivity of TRAIL-induced HCC cell apoptosis.
Similarly, previous studies have shown that the apoptotic inhibitors of cIAP2 and survivin are important factors in determining the apoptosis of HCC cells (3,14).Thus, we investigated the expression of the two proteins.The results showed that cIAP2 and survivin were downregulated after silencing SNAIL.Additionally, we found that a decrease of survivin expression not only induced HCC cell apoptosis augment, but also enhanced the sensitivity of TRAIL-mediated apoptosis in HCC cells following the knockdown of SNAIL.However, cIAP2 did not affect TRAIL-induced apoptosis.Nevertheless, the exact mechanism on how these proteins are regulated by SNAIL silencing remains to be clarified.
A previous study demonstrated the crucial role of SNAIL in the resistance of tumor cells to TRAIL (41).As a downstream target of TRAIL, many factors acted on this role of SNAIL.Furthermore, our results show that, SNAIL is important in mediating these factors, exerting an indirect effect.SNAIL has been shown to suppress Raf-kinase inhibitor protein (RKIP) transcription and expression, which was consistent with our observation that Raf-1 protein was downregulated after SNAIL silencing, and previous findings have shown that RKIP inhibited NF-κB activity (42,43).Another study showed that NF-κB induces the anti-apoptotic regulator Mcl-1, which was able to cause TRAIL-resistance for cancer (13).Therefore, the potential mechanism was that silencing SNAIL could indirectly decrease Mcl-1 to mediate the sensitization of TRAIL-induced apoptosis in HCC cells by upregulating RKIP and downregulating NF-κB.Thus, silencing SNAIL downregulated Raf protein to influence NF-κB activity, thereby sensitizing HCC cells to TRAIL, which has been confirmed in our study.Nevertheless, the mechanisms involved remain to be elucidated.
In conclusion, SNAIL is a vital mediator of HCC cell sensitivity to TRAIL-induced apoptosis.Inhibition of SNAIL through shRNA carried by lentivirus is an important method that reversed HCC cell resistance to TRAIL by affecting the NF-κB pathway.Therefore, SNAIL can be a significant gene for treating HCC and identification of a new target to change the sensitization of TRAIL-induced apoptosis in HCC is an important therapeutic strategy.
Figure 1 .
Figure 1.Characterization of LV-shSNAIL expression.SNAIL protein expression was detected prior to and after cell infection with LV-shSNAIL by western blotting using anti-SNAIL in three cell lines.Quantitative PCR was used to analyze mRNA expression level of SNAIL following infection with LV-shSNAIL in the three cell lines and control groups.The expression level of SNAIL in (A) HepG2, (B) SMMC7721 and (C) HuH-7 cells.** P<0.05, compared with either Mock or shCON.
Figure 3 .
Figure 3.Effect of SNAIL knockdown and addition of TRAIL to different groups of HCC cell lines on cell proliferation.HepG2, SMMC7721 and HuH-7 hepatocellular carcinoma cells were divided into four groups and infected with Mock, LV-shSNAIL, Ad5.TRAIL and LV-shSNAIL + Ad5.TRAIL, respectively.Cell viability was examined by MTT assay at different time points after infection.Four group reagents were added separately to (A) HepG2, (B) SMMC7721 and (C) HuH-7 cells.** P<0.05, each group compared with Mock.
Figure 4 .
Figure 4. Knockdown of SNAIL and addition of TRAIL-induced apoptosis in the HepG2, SMMC7721 and HuH-7 cell lines.Cells were divided into four groups and incubated in Mock, LV-shSNAIL, Ad5.TRAIL and LV-shSNAIL + Ad5.TRAIL, respectively.The cells were stained with Hoechst 33258 for 48 h and analyzed using a fluoresence microscope.** P<0.05, each group compared with Mock. | 2018-04-03T01:06:56.285Z | 2015-03-01T00:00:00.000 | {
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7499460 | pes2o/s2orc | v3-fos-license | Analysis of aggregate exposure to chlorpyrifos in the NHEXAS-Maryland investigation.
As part of the National Human Exposure Assessment Survey (NHEXAS) in Maryland, we collected indoor air, carpet dust, exterior soil, and duplicate diet samples from a stratified random sample of 80 individuals to evaluate aggregate daily exposure, contributions of specific pathways of exposure, and temporal variation in exposure to chlorpyrifos. We collected samples from each participant in up to six equally spaced sampling cycles over a year and analyzed them for chlorpyrifos. We used chlorpyrifos concentrations in each medium and self-reported rates of time spent inside at home, time and frequency of contact with carpet, frequency of contact with soil, and weights of the duplicate diet samples to derive exposure to chlorpyrifos from each medium as well as average daily aggregate exposure (nanograms per day). The mean aggregate daily exposure to chlorpyrifos of 36 measurements obtained from 31 people was 1,390 ng/day (SD, 2,770 ng/day). Exposure from inhalation of indoor air accounted for 84.7% of aggregate daily exposure to chlorpyrifos on average. Chlorpyrifos concentrations in indoor air and carpet dust and the corresponding exposure rates were significantly correlated. Repeated short-term measurements of chlorpyrifos in carpet dust from individual residences were strongly correlated over time (reliability coefficient, R = 0.90), whereas the short-term measurements of chlorpyrifos in indoor air (R = 0.55) and solid food (R = 0.03) had moderate to weak reliability. Exposure to chlorpyrifos through those media and in aggregate based on direct measurements reported in this study can be used to understand better the accuracy of pesticide safety assessments.
Passed into law in 1996, the U.S. Food Quality Protection Act (FQPA) requires a more comprehensive assessment than ever before of pesticide exposure, dose, and effects (1,2). In particular, the FQPA directs the U.S. Environmental Protection Agency (EPA) to consider exposure to potentially sensitive subgroups in the population, coincident dietary and nondietary (i.e., aggregate) exposure, and contemporaneous multichemical (i.e., cumulative) exposure for pesticide risk assessment. To implement the FQPA, the U.S. EPA has been developing new methods and models to assess aggregate pesticide exposure that could occur in community settings (3)(4)(5). Few reports have been published on direct measurements of pesticide exposure from different potential exposure pathways and the aggregate exposure posed by these exposures in residential settings. This information would be valuable for evaluating exposure models and for epidemiologic studies of the relationship between personal pesticide exposure and possible human health effects (6)(7)(8)(9).
In this paper we report the results of a longitudinal study of aggregate daily exposure to chlorpyrifos, a commonly used organophosphate pesticide, in community and agricultural settings. The data presented are the product of a pilot investigation of temporal variation in human exposure to selected contaminants in multiple mediathe National Human Exposure Assessment Survey in Maryland (NHEXAS-MD). The objectives of our study were to assess aggregate daily exposure to chlorpyrifos from indoor air, carpet dust, exterior soil, and food; to identify the predominant pathways of chlorpyrifos exposure among those media; and to evaluate the reliability of a short-term measure of exposure for assessment of longterm average chlorpyrifos exposure.
Methodology
Study population. A stratified probability sample of 80 individuals over 10 years of age selected from four contiguous counties and the city of Baltimore in Maryland enrolled in the study from September 1995 to September 1996. All participants provided informed consent under protocols approved by an institutional review board. Details of the sampling strategy and demographic characteristics of the participants are reported elsewhere (10). Briefly, we collected samples from selected environmental and biologic media, as well as questionnaire data, from each participant in as many as six 1-week periods (cycles) approximately equally spaced between September 1995 and September 1996. Cycles 1-6 correspond to 20 September to 23 December 1995, 15 January to 23 February 1996, 27 March to 20 April 1996, 22 April to 15 June 1996, 18 June to 27 July 1996, and 30 July to 18 September 1996, respectively.
Sample collection and analysis. We collected an indoor air sample by using a small pump to draw air through an integrated sampler containing an inertial impactor with a particle cut-point of 10 µm followed by a filter and polyurethane foam (PUF) plug (URG Inc., Chapel Hill, NC). We placed the sampler approximately 1.5 m above floor level in an area of unrestricted air flow in the principal activity room of the household as identified by the study participant. The pump ran at 4 L/min with a programmable timer-controller that directed air through the PUF sampler for 10 min out of each 70-min period during 1 week. Total collection time for each sample was 24 hr, and the target sample volume was 5.76 m 3 . Participants completed a questionnaire on daily time budgets and behavior patterns on each day of the 7-day sampling period for a given cycle. We used responses to questions concerning body weight and time spent inside at home to estimate the inhalation rate and average daily time inside at home, respectively, for each participant.
We obtained a house dust sample on the first day of each sampling period by vacuuming the carpet in the activity room of the household using a high-volume small surface sampler (HVS3; CS-3, Inc., Sandpoint, ID) (11). By making eight passes with the nozzle over each strip of carpet, we collected house dust > 5 µm in diameter into a precleaned Teflon bottle with size selection effected by a cyclone separator. We sieved out particles > 150 µm in diameter in the laboratory before extraction and analysis of the dust samples. We recorded the area sampled, which had a target value of 2 m 2 . Responses to the question, "How much time did you spend laying down or sitting on the carpet or rugs at home today?" gave us the average daily frequency of contact with carpet and average daily time on carpet.
Where possible, we obtained a soil sample from the respondent's residence on the first day of each home visit. We took soil samples from the yard to evaluate potential exposure from bare soil and play areas. If the household had a garden for growing food, then we obtained a sample of garden soil to evaluate this possible food and dermal exposure pathway. We sampled foundation soil to evaluate the potential for exposure from past application of pesticides for termite control or other uses. If we found no areas of the property that met the sampling criteria, we took no samples. We composited aliquots of soil obtained from different locations of the residence into a single sample in the field, and used responses to the question, "Did you have soil or dirt from your yard in contact with the skin today?" to obtain average daily frequency of contact with soil.
Because of limited resources, we selected a portion of the indoor air, carpet dust, and soil samples for analysis. We analyzed approximately 75% of the indoor air and carpet dust samples collected in cycles 1, 3, and 5, and approximately 25% of those sample types collected in cycles 2, 4, and 6. To conserve resources further, we analyzed approximately half as many soil samples as indoor air or carpet dust samples based on the assumption that this outdoor exposure medium was less important and less variable than indoor exposure media in this population. Details of sample selection criteria can be obtained from the authors. We analyzed indoor air, carpet dust, and soil samples for chlorpyrifos and 10 other pesticides at Southwest Research Institute in San Antonio, Texas. Briefly, samples were Soxhlet extracted with 6% ethyl ether in hexane, cleaned through a Florisil column, and analyzed by gas chromatograph/mass spectrometry-selected ion monitoring with a 30 m × 0.25 mm i.d. DB5 column (J & W Scientific, Folsom, CA).
Details of the solid food sample collection, analysis, and diet questionnaire procedures are reported elsewhere (9,12). Briefly, we requested participants to prepare a duplicate portion of meals consumed on four consecutive days during each sampling cycle. A field technician recorded the weight of each 4-day solid food sample in cycles 2-6. We homogenized samples (solid food separate from beverages) and analyzed them for selected pesticides at the U.S. Food and Drug Administration laboratory in Kansas City, Missouri. We did not record the weights of duplicate solid samples in cycle 1; hence, we used the average weight of duplicate solid samples on cycles 2-6 for each respondent to estimate the respondent-specific duplicate diet weights in cycle 1.
Quality assurance. To ensure traceability and accuracy of the data, we performed a series of quality assurance steps. A chain-ofcustody form followed each sample and questionnaire from the field to the laboratory and finally to the database manager. We omitted from subsequent analysis any sample data point not accompanied by a completed chain of custody. We analyzed field blanks, duplicate field samples, and reagent blanks for the presence of chlorpyrifos as quality control measures of field and laboratory methods. We did not detect chlorpyrifos in any field blank or reagent blank in our study. Chlorpyrifos concentrations were comparable in the pairs of primary and duplicate samples. We determined detection limits (DL) and recovery efficiencies for chlorpyrifos in each medium throughout the study. The average DL was 0.720 ng/m 3 (range, 0.577-0.773 ng/m 3) in indoor air samples, 240 ng/g (35.2-1,700 ng/g) in carpet dust samples, and 5.24 ng/g (2.96-9.16 ng/g) in soil samples. The chlorpyrifos DL in food samples was 100 ng/kg and was constant over the course of the study. We determined recovery efficiency by fortified samples. We spiked extraction matrices with known amounts of analyte, which were about 200 ng of chlorpyrifos into PUF and 6 µg of chlorpyrifos into 2.0 g of sieved dust or 30 g of soil, and we spiked solid food samples to a concentration in the range of 10.9-18.6 µg/kg. We analyzed spiked samples as ordinary samples. The spike recoveries centered near 100% and had a range of 95.0-119% for PUF samples, 78.0-152% for dust samples, 92.0-144% for soil samples, and 84.7-95.8% for duplicate solid food samples. Data analysis. The models, variables, and constants in the data set used to estimate exposure to chlorpyrifos from each pathway are shown in Table 1. Chlorpyrifos concentrations in each medium below the respective DL were set to zero. We quantified inhalation of indoor air, incidental ingestion of carpet dust, incidental ingestion of soil, dermal absorption of carpet dust, dermal absorption of soil, and ingestion of food as the pathways of exposure to chlorpyrifos. We calculated pathway-specific exposure (nanograms per day) as a function of chlorpyrifos concentration in the exposure medium; time spent in the microenvironment; inhalation, ingestion, or contact rate with the medium of interest; and the fraction of chlorpyrifos absorbed by the lung, skin, and gastrointestinal tract (13). We computed aggregate daily exposure to chlorpyrifos as the sum of average daily exposure from all six pathways.
We generated descriptive statistics for chlorpyrifos concentration and exposure from each pathway and in aggregate, samples containing a detectable amount of the analyte. We computed contributions to aggregate exposure from each pathway for each observation. The data exhibited strong positive skewness, and some exposures were the product of binary factors yielding nonnormal distributions (skewness > 3.21). For nonzero pairs of data, we used Spearman correlation to evaluate rank associations between chlorpyrifos concentrations in sample media and exposures from different pathways. We calculated Pearson correlation coefficients for comparison as well.
Reliability is a concept used to describe the degree to which a randomly selected single measure of exposure taken from a set of measures for an individual represents their long-term average exposure. To estimate the reliability of a short-term measure of daily exposure to chlorpyrifos for individuals, we computed the intraclass correlation coefficient of reliability (R) with indoor air, carpet dust, and food concentration data. R is the ratio of between-person variance to the total variance observed in a repeated-measure study (14). R ranges from 0 to 1, with values near zero indicating low reliability and values near one indicating high reliability. In this study, the temporal variability observed is a characterization of within-person variability, whereas the total variability is a combination of the temporal variability of within-person and the between-person variability.
Results
The final data set contained 107 observations from 44 participants for indoor air, 126 observations from 50 participants for carpet dust, 60 observations from 41 participants for soil, and 379 observations from 75 participants for solid food. Thirty-six home visits from 31 participants yielded contemporaneous measurements of indoor air, carpet dust, soil, and solid food, and we used these observations to calculate aggregate daily chlorpyrifos exposure. The numbers of observations for each medium of the six sampling cycles are shown in Table 2.
Chlorpyrifos was present at detectable levels in 92.5% of indoor air samples, 79.4% of carpet dust samples, 40% of soil samples, and 38.3% of solid food samples. Table 3 presents summary statistics of chlorpyrifos concentrations in indoor air, carpet dust, soil and solid food samples; exposure to chlorpyrifos from each pathway; and aggregate daily exposure. The mean dust loading from participating households was 3.55 g/m 2 (SD, 8.01 g/m 2 ) with a range of 0.10-51.97 g/m 2 . The distribution of chlorpyrifos concentrations in each medium was skewed right and ranged over two to four orders of magnitude. The fraction of sampling visits for which there was nonzero exposure to chlorpyrifos because of detectable levels of chlorpyrifos in the exposure medium and contact with the exposure medium were as follows: indoor air, 93% (99 of 107); carpet dust, 45% (57 of 126); soil, 18% (11 of 60); and food, 38% (135 of 356). Aggregate daily chlorpyrifos exposure computed as the sum of exposure from the six pathways was also skewed right and ranged from 13.5 ng/day to 12,800 ng/day, with a mean of 1,390 ng/day (SD, 2,770 ng/day).
Exposure from indoor air accounted for the majority of aggregate daily exposure to chlorpyrifos, contributing 84.7% on average. Solid food intake accounted for 13.2% of the average aggregate daily exposure. Incidental ingestion of carpet dust, dermal absorption of carpet dust, incidental ingestion of soil, and dermal absorption of soil contributed 0.06%, 0.76%, 1.18%, and 0.01%, respectively, on average to aggregate daily exposure. The percentage contributions to aggregate chlorpyrifos exposure from each pathway for each observation are shown in Figure 1. Exposure from inhalation of indoor air accounted for the majority of aggregate exposure for most observations. In several high-aggregate-exposure observations, contributions of solid food accounted for a substantial fraction of the total.
We restricted correlation analysis to those observations with nonzero measurements and between pathways that we considered a priori We calculated reliability to evaluate temporal variability in the data sets restricted to participants who were involved in two or more sampling cycles for indoor air, carpet dust, and solid food. The intraclass correlation coefficient of reliability was 0.55 for the concentrations of chlorpyrifos in indoor air (n = 85). For the concentrations of chlorpyrifos in carpet dust (n = 99) and duplicate plates (n = 356), the intraclass correlation coefficient of reliability was 0.90 and 0.03, respectively.
Discussion
Information on the sources and magnitude of chlorpyrifos exposure is important for exposure and risk assessment of populations and individuals regarding potential health impacts of this common insecticide. In addition, the direct measurements of exposure to chlorpyrifos through indoor air, carpet dust, soil, and solid food reported in this study can be used to understand better the accuracy of pesticide safety assessments based on indirect methods or models.
Chlorpyrifos concentrations and exposures through different pathways based on those media have been measured or modeled in other studies, although few of these investigations contain the breadth of exposure media and pathways provided through the present work. The chlorpyrifos detection frequency and concentrations in our study for indoor air, carpet dust, and soil were in the same range as those from NHEXAS-Arizona (15) and the Minnesota Children's Pesticide Exposure Study (MNCPES) (16). Chlorpyrifos concentrations in settled dust and indoor air following indoor broadcast and other application methods may be 10-fold greater than the corresponding concentrations in the present study (17)(18)(19). Yet, our highest levels are in the range of the lowest levels observed up to 10 days after indoor application of chlorpyrifos (18).
Scenario-based estimates of exposure to chlorpyrifos are much greater than the exposures observed in our study and sometimes produce different conclusions about the relative contributions from different exposure media. For example, Fenske et al. (17) concluded that dermal absorption represented approximately 68% of the aggregate exposure (0.04-0.06 mg/kg/day) to a hypothetical infant. In contrast, our results agreed with Byrne et al.'s (18) finding that contact with household surfaces and subsequent hand-tomouth activity contribute little to overall chlorpyrifos exposure. Similarly, estimates of nondietary ingestion of chlorpyrifos from treated indoor and outdoor surfaces published in a modeling study were up to several orders of magnitude higher than corresponding measures in our study (5). Several investigations have been conducted of exposure to chlorpyrifos via solid food ingestion. Details of comparisons between those works and our study are reported elsewhere (9). Caution should be exercised when comparing exposure values across studies because of differences in application rates, study populations, and sampling and analysis protocols.
To evaluate aggregate daily exposure to chlorpyrifos accurately, pathways and activities that represent the greatest potential exposure should be identified correctly. Aggregate daily exposure as assessed in the present study accounted only for pathways and activities related to the residence. Exposure to chlorpyrifos from environmental media in work areas and other places is required to conduct a more comprehensive aggregate assessment. Nevertheless, the time activity data show that people in this population spent the majority of their time inside at home every day (mean = 16 hr), which suggests residential exposure is an important part of aggregate exposure. The duplicate plate methodology employed here is prone to its own types of errors. For example, 9% of the respondents indicated that at least some food was not included in the diet samples for reasons including illness, travel, not eating at home, limited food availability, and fatigue. Regarding limitations of the inhalation assessments, we measured chlorpyrifos concentrations in indoor air in one location in each household. Data are needed on the spatial distribution of indoor air chlorpyrifos levels to assess the impact of this limitation on our results, although data for other air pollutants indicate that spatial variation within homes is low in the absence of discrete emission sources (20,21). Similarly, we collected dust samples from a limited area of carpet in each household. Additional data are needed to evaluate the spatial variability of chlorpyrifos in settled dust indoors including carpet and hard surfaces in the household. However, data from the MNCPES indicate that chlorpyrifos loading (nanograms per square centimeter) on carpet and smooth indoors surface may be similar (16); thus, we did not quantify a potentially significant pathway of dermal and incidental ingestion exposure. We did not detect chlorpyrifos residues in duplicate beverage and drinking water samples (9,22); thus, omitting these media from the assessment had no impact on estimates of aggregate exposure. Our ability to evaluate soil-derived exposure was limited by the small sample size for this medium (only 60 observations from three cycles). Although the available information suggests soil is a minor contributor to aggregate exposure for this population, more data are needed to assess this pathway fully. We obtained contact frequency and contact time for all pathways except ingestion from food in our study from questionnaires rather than via direct measures based on observations, videography, or even diaries (23). That means that our results may be influenced by participants' memories. We obtained factors used to estimate inhalation rate, transfer of dust and soil from surface to skin, and absorption of chlorpyrifos from the exposure literature (Table 1). For example, we estimated the adherence factor used in dermal absorption of soil as the average of the adherence factors associated with potentially relevant outdoor activities (13). Because of these limitations in knowledge, the assessment conducted for dermal and incidental ingestion exposure is highly uncertain.
A principal objective of this study was to identify the important pathways of chlorpyrifos exposure for this population. We found that inhalation of indoor air and ingestion of solid food accounted for almost all (97.9% together) exposure to chlorpyrifos on average. This result is based on only 36 observations for which we have contemporaneous chlorpyrifos concentration and exposure factor data for indoor air, carpet dust, soil, and solid food. By omitting soil pathways, 96 observations of aggregate exposure are obtained based on indoor air, carpet dust, and solid food. In that case, exposure from inhalation of indoor air still accounted for the majority (76.1%) of aggregate daily exposure to chlorpyrifos on average, followed by solid food intake at 22.8%. Incidental ingestion and dermal absorption of carpet dust contributed, on average, 0.04% and 1.0%, respectively, in this larger data set. This information indicates that our conclusions about contributions from each pathway were not unique to the 36-observation data set.
Our choice to treat nondetectable chlorpyrifos concentrations as zero rather than a nonzero value did not bias the aggregate exposure findings. For example, when we set nondetects to one-half the DL for the respective media, inhalation accounted for 72% of population exposure and diet for 26%, and each of the other pathways contributed < 1% to the total exposure.
Lack of knowledge about absorption of chlorpyrifos in the lung and gastrointestinal tract and through the skin contributes to uncertainty in the exposure estimates presented here. For example, we are not aware of empirical data on chlorpyrifos absorption following respiratory exposure. We assumed that 100% of inhaled chlorpyrifos is absorbed, following Hubal et al. (4) and Byrne et al. (18), whereas other investigators have assumed a 70% absorption efficiency for respiratory exposures (24,25). Human volunteers who ingested neat chlorpyrifos are estimated to have absorbed 70-90% of the administered dose (26,27). We assumed that 50% of ingested chlorpyrifos is absorbed in accordance with assumptions made by a team of U.S. EPA investigators (4) under the assumption that the food matrix inhibits absorption. The dermal absorption efficiency of chlorpyrifos is reported to be approximately 1% based on studies with human volunteers (26,27). Values in this range have been used in other dermal exposure and dose-modeling studies (4,18,25). Additional knowledge is needed about absorption of chlorpyrifos across biologic membranes. Yet, the current degree of uncertainty does not alter our findings that inhalation and dietary ingestion are the principal pathways of exposure for this study population.
The findings from repeated-measure studies have implications for tools such as epidemiology and quantitative risk assessment that are used to evaluate the potential effects of environmental contaminants on human health. In the NHEXAS-MD study, we found moderate (R = 0.55) within-person variability of chlorpyrifos concentration in indoor air over time, indicating that withinperson and between-person variation contributed almost equally to total variance in the short-term measure of indoor air concentration. We found low temporal variability of chlorpyrifos concentration in carpet dust, indicating that the timing of dust sample collection may not be an important design consideration in the absence of a recent pesticide application event. But mean time spent inside the home and carpet contact rate varied significantly among cycles and among days for individuals (10,28). Dietary intake of chlorpyrifos exhibited low reliability, perhaps because of variation in shortterm food consumption and in the occurrence and exposure concentration of chlorpyrifos among servings of food commodities. Thus, with regard to determining the levels of exposure for an epidemiologic study, our results indicate that a single shortterm measure of chlorpyrifos exposure for an individual based on environmental monitoring and exposure factor data may not yield an accurate estimate of chronic exposure for that individual.
Conclusion
Research is needed to reduce uncertainty about exposure concentrations, factors, and models and to yield more realistic assessments of aggregate exposure to environmental contaminants (4). NHEXAS-MD is a pilot investigation for future national-scale, multimedia, multipollutant exposure assessment studies. The study design affords evaluation of aggregate daily chlorpyrifos exposure from realistic ordinary life in a randomly sampled population. Direct measurements of chlorpyrifos concentrations in potential contact media and time activity pattern data such as those reported here, in conjunction with data from controlled experiments and improved exposure factor information, can help to reduce uncertainty in pesticide exposure and risk assessments. The results of the present aggregate exposure study are based on measurements of chlorpyrifos in indoor air, carpet dust, soil, and solid food. The aggregate exposure rate for chlorpyrifos varied over a wide range and up to 12.8 µg/day. Inhalation of indoor air was the most important pathway of aggregate exposure. Concentrations of chlorpyrifos in indoor air and carpet dust were significantly correlated, which may have implications for aggregate exposure to this substance and other semivolatile insecticides. The NHEXAS-MD study design helps to illustrate patterns in aggregate temporal exposure to chlorpyrifos through various media. The timing of sample collection may not be important for assessment of chlorpyrifos concentration in carpet dust but could influence the results of indoor air and solid food chlorpyrifos measurements. However, variation in exposure to chlorpyrifos from those media and in aggregate should be considered with respect to temporal variability of people's activity patterns. | 2014-10-01T00:00:00.000Z | 2002-03-01T00:00:00.000 | {
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26534025 | pes2o/s2orc | v3-fos-license | Nuclear Protein in Testis Midline Carcinoma Presenting in an Infant as a Pericardial Mass with Staging by 18 F‑Fluorodeoxyglucose‑positron Emission Tomography/Computed Tomography
Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare, aggressive, lethal, epithelioid, poorly differentiated cancer first described in Japan in 1991, unique in that is defined genetically rather than by histological tissue of origin. It usually arises in the body midline and presents as a mass with metastasis. An infant presenting with pneumonia was found to have a pericardial mass, NMC resected, and subsequent staging positron emission tomography (PET) showing residual mediastinal tumor and midline abdominal metastases. Fewer than 100 cases of NMC have been reported in the literature, and PET appears to be the imaging modality of choice in complete staging and evaluation of treatment response.
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com mass with small pericardial effusion. CT [ Figure 2] and magnetic resonance imaging (MRI) [ Figure 3] showed a 7.3 cm × 3.6 cm mass adjacent to the left ventricle with both soft tissue and fluid components exerting a mass effect on the left ventricular chamber. A median sternotomy was performed with 95% resection/cytoreduction of the mass. Fluorescence in situ hybridization revealed t(15;19) mutation and immunohistochemistry exhibited diffuse nuclear reactivity for NUT; histopathology showed high-grade malignancy composed of primitive-appearing round to ovoid-shaped cells, with irregular nuclei and scant pale eosinophilic cytoplasm arranged in a vague nested growth pattern with areas of myxoid change, focal-spindled morphology, and large areas of necrosis, consistent with NMC. Two weeks after subtotal surgical resection, FDG-PET/CT [ revealed increased uptake in a 2.7 cm × 2.3 cm pericardial soft tissue density abutting the superior aspect of the left ventricular base, maximum standardized uptake value (SUV) 2.5; midline abdominal mesenteric soft tissue tumor, SUV 4.5; no metastasis to solid organs, bone, or lung parenchyma.
Following subtotal resection of the primary pericardial tumor, the patient was started on systemic chemotherapy with paclitaxel, ifosfamide, cisplatin, and vorinostat while awaiting the US Food and Drug Administration approval for compassionate use of a small molecule bromodomain and extraterminal motif (BET) inhibitor. Nine weeks postoperative, an FDG-PET/CT [ Figures 8 and 9] showed disease progression while on the BET inhibitor, and she received multiple other standard chemotherapy regimens, including cyclophosphamide, doxorubicin, vincristine, topotecan, irinotecan, and temozolomide. Despite initial stabilization of disease, she ultimately experienced disease progression and died approximately 11 months following diagnosis. The true incidence of NMC is unknown as it is often confused with other SCCA arising in the aerodigestive tract. In young adults (median age 32.5 years), 11 of 98 lethal carcinomas were NMCs, median age 17.6 years, range from 0.1 to 78 years. [2] Cases have been reported in all ages and both sexes.
The mechanism of action of the BRD-NUT protein is not well understood; however, the BRD4 is known to bind to acetylated histones and is thought to preserve cellular memory by marking these regions for transcription. NUT is expressed in postmeiotic spermatids and has unknown function. [3] The BRD-NUT protein may modify chromatin to block cell differentiation, allowing progenitor cells to continue proliferating.
CT with intravenous contrast is standard for initial staging, with NMC typically appearing as a hypoattenuating heterogeneously enhancing infiltrative mass with poorly defined margins and internal necrosis, with local invasion. Adjunctive MRI is performed if there is a concern for the chest wall, vascular, or cardiac invasion. [4] The signal intensity on T2-weighted MRIs is that of a cellular neoplasm, but imaging characteristics are otherwise indistinguishable from other high-grade neoplasms such as lymphoma or sarcoma. [5] FDG-PET/CT is the preferred modality for guiding biopsy to viable tissue, staging and assessment of metastatic disease, evaluation of disease treatment response, and restaging with assessment of disease extent and severity over time. [4] NMC has a poor clinical response to aggressive chemotherapy and radiation, and to date, there has been only one known case of successful treatment of a 10-year-old boy with NMC of the iliac bone. [6] Therapeutic targeting of BRD4-NUT fusion proteins by inhibitors targeting the bromodomain of BRD4 (BET inhibitors) leads to dissociation of BRD4-NUT from the MYC transcription promoter, leading to cellular apoptosis. Several phase 1 and one phase 2 clinical trials are underway evaluating BET inhibitors for the treatment of hematologic malignancies, lymphoma, metastatic castration-resistant prostate carcinoma, and advanced solid tumors, as well as NMC. [7,8] An International NMC registry has been established. [9] NMC, a poorly differentiated highly aggressive malignancy with no specific histological tissue of origin, is likely underdiagnosed, rapidly lethal, mostly occurs in the midline above the diaphragm, and is in need of targeted therapy. We present a case of NMC in an infant, in the midline above and below the diaphragm as staged by FDG-PET/CT, responding poorly to chemotherapy.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest. | 2018-04-03T00:45:02.255Z | 2017-07-01T00:00:00.000 | {
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232092517 | pes2o/s2orc | v3-fos-license | LEAF: Simulating Large Energy-Aware Fog Computing Environments
Despite constant improvements in efficiency, today’s data centers and networks consume enormous amounts of energy and this demand is expected to rise even further. An important research question is whether and how fog computing can curb this trend. As real-life deployments of fog infrastructure are still rare, a significant part of research relies on simulations. However, existing power models usually only target particular components such as compute nodes or battery-constrained edge devices.Combining analytical and discrete-event modeling, we develop a holistic but granular energy consumption model that can determine the power usage of compute nodes as well as network traffic and applications over time. Simulations can incorporate thousands of devices that execute complex application graphs on a distributed, heterogeneous, and resource-constrained infrastructure. We evaluated our publicly available prototype LEAF within a smart city traffic scenario, demonstrating that it enables research on energy-conserving fog computing architectures and can be used to assess dynamic task placement strategies and other energy-saving mechanisms.
Abstract-Despite constant improvements in efficiency, today's data centers and networks consume enormous amounts of energy and this demand is expected to rise even further. An important research question is whether and how fog computing can curb this trend. As real-life deployments of fog infrastructure are still rare, a significant part of research relies on simulations. However, existing power models usually only target particular components such as compute nodes or battery-constrained edge devices.
Combining analytical and discrete-event modeling, we develop a holistic but granular energy consumption model that can determine the power usage of compute nodes as well as network traffic and applications over time. Simulations can incorporate thousands of devices that execute complex application graphs on a distributed, heterogeneous, and resource-constrained infrastructure. We evaluated our publicly available prototype LEAF within a smart city traffic scenario, demonstrating that it enables research on energy-conserving fog computing architectures and can be used to assess dynamic task placement strategies and other energy-saving mechanisms.
Index Terms-Simulation, Modeling, Fog Computing, Edge Computing, Energy Consumption
I. INTRODUCTION
With the rise of the internet of things and the beginning of the fourth industrial revolution, many sectors and environments are expected to become interspersed with connected sensors. As these sensors produce an ever-increasing amount of data, the classical cloud computing approach of sending all information directly to a few powerful data centers, often far away from the data source, is not feasible anymore. One attempt to solve these emerging problems is to deploy a distributed layer of compute nodes close to the edge of the network that filters, aggregates, and preprocesses the raw data generated by end devices and sensors. This new paradigm, called fog computing, promises to save large amounts of bandwidth, reduce latencies for time-sensitive applications, and increase end-user privacy as the fog nodes can take care of aggregating and anonymizing data [1]. However, due to their distributed and heterogeneous nature, fog computing environments are much more challenging to manage than centralized data centers [2].
At the same time, energy consumption of information and communications technology is already accounting for more than 10 % of global energy consumption and is expected to exceed the 20 % mark by 2030 [3]. An open research question is whether fog computing will be able to reduce the overall energy consumption of future computing infrastructure or if it may even be responsible for a further increase [1], [4], [5].
Although the general trend of concentrating computing resources into hyper-scale data centers continues [6], research has shown that distributed architectures could save between 14 % to more than 80 % of total energy consumption compared to fully centralized architectures [7], [8].
A major part of today's fog computing research is centered around modeling and simulations. Nevertheless, to the best of our knowledge, there exists no tool that is suitable to explore the overall power usage of large-scale fog deployments and enables research on energy-conserving architectures and algorithms in this domain. Major existing fog computing simulators provide no energy modeling at all [9]- [11] or only focus on specific components of fog infrastructures [12], [13]. Furthermore, many simulators fail to realistically model fog environments by leaving out substantial aspects like mobility [10], [12]. Existing analytical approaches on the other hand provide only a high level of abstraction and cannot represent change over time, prohibiting the modeling of mobility and evaluation of online decision making algorithms [4], [7], [14].
We present LEAF 1 , a new simulator for modeling Large Energy-Aware Fog computing environments. In contrast to the related work, our model was designed with the following four requirements in mind: (1) Realistic Fog Computing Environment: Enable the simulation of applications with complex task graphs in distributed, heterogeneous, and resource-constrained fog computing environments. Nodes can be mobile and they can join or leave the topology at any time. (2) Holistic Energy Consumption Model: Enable the separate assessment of power usage of compute nodes, network traffic, and applications, allowing for the creation of load profiles for individual parts of fog computing environments. (3) Energy-Aware Online Decision Making: Enable research on task placement strategies, routing policies, and energy-saving mechanisms that dynamically adapt their behavior based on the power usage of infrastructure and applications. (4) Performance and Scalability: Enable the simulation of complex scenarios with thousands of devices and applications at least two orders of magnitude faster than real-time on commodity hardware.
Our model combines analytical and discrete-event modeling to enable easy-to-analyze large-scale experiments. Specifically, we model infrastructures and applications as variable graphs that can change throughout the simulation and do not rely on representing individual packets. This way, the model enables research on novel task placement strategies or energy-saving mechanisms that must be evaluated in large settings. We expect LEAF to help both researchers and practitioners assess the power usage characteristics of different cloud, fog, and edge computing architectures, which can lead to more well-founded decisions when planning future infrastructures.
The remainder of the paper is structured as follows. Section II discusses the related work. Section III introduces our infrastructure and application model for fog computing environments. Section IV explains the different kinds of power models that can be applied. Section V evaluates our model on a smart city scenario. Section VI concludes the paper.
II. RELATED WORK
Energy-efficient data centers have been a research area for years as electricity costs account for a significant part of a data center's operating expenditure. In fog or edge computing, however, most research on power usage only concerns specific components such as battery-constrained edge devices [13] or fog nodes [12]. The bigger picture, namely the potential impact of fog computing on the overall energy consumption of information and communications technology, has retrieved comparably little attention.
One approach to assess the estimated consumption of fog computing environments is analytical modeling. These models are usually suitable for examining large-scale scenarios since they are fast to execute and convenient to experiment with. Sarkar et al. [14] compare service latency and energy consumption of fog and cloud computing by performing a high-level examination of different architectures. The authors focus on infrastructure only and incorporate no concept for applications. Furthermore, they do not distinguish between static and dynamic energy consumption, which limits the informative value of the analyses. Jalali et al. [4] model energy usage by using a flow-based model for highly shared equipment such as core routers and switches, and a time-based model for enduser equipment that is usually not shared among many clients. Their findings show that the power impact of fog computing depends on many factors such as the type of attached access network. However their scenario is a relatively specific use case and not suitable as a general purpose model. Ahvar et al. [7] propose a model that differentiates between static and dynamic power usage and incorporates the consumption of network devices and cooling. Even though their model is relatively comprehensive, it incorporates several critical simplifications such as an even distribution of VMs among data centers and the assumption that all fog nodes connect to the same number of devices. Moreover, there exists no concept of applications or tasks, nor is there a way to calculate the relative power requirements of VMs. All presented analytical approaches suffer from the fact that they model at a high level of abstraction and, for example, make the invalid assumption that fog nodes are homogeneous [7], [14]. Moreover, they cannot represent change over time, prohibiting the assessment of mobility and online decision making algorithms.
Besides analytical models, a large number of discrete-event simulators has been developed in recent years. Although there exist cloud computing simulators that feature comprehensive power models like GreenCloud [15], these tools are not designed to model highly heterogeneous and variable environments like those encountered in fog computing.
Many popular simulators in the domain of fog and edge computing such as EdgeCloudSim [9] or IOTSim [10] comprise no power modeling at all. Others, like YAFS [11], allow users to specify custom attributes related to node power usage but do not feature any power modeling at runtime. The simulators that do are either overly simplistic or focus only on specific components of the infrastructure. For example, iFogSim [12] inherits some power modeling capabilities from CloudSim and can model the power consumption of data centers and fog nodes. Nevertheless, there is currently no way to simulate the power usage of edge devices, networks, or applications. PureEdgeSim [13] features a comprehensive energy consumption model for edge devices, including Wi-Fi traffic, to simulate battery-constrained devices. However, the consumption of data centers, fog nodes or wide area network (WAN) traffic cannot be modeled. FogNetSim++ [16] is the only fog computing simulator to our knowledge that features an energy model for applications. Albeit the focus on performance, the detailed modeling of network traffic on a per packet basis inevitably results in worse scalability than our model. Furthermore, due to our analytical approach, it is a lot easier to set up experiments in LEAF, explore different configurations, and analyze results.
III. FOG ENVIRONMENT MODEL
The way LEAF models fog computing environments is inspired by analytical modeling. Infrastructure and applications are represented as graphs and power models as functions that operate on these graphs. Together, these components define the state of the simulation at a certain time step, which we Fig. 1: LEAF uses discrete events to update the state (configuration) of the simulation. In this example, an additional compute node (a car) was added to the infrastructure graph and a new application was placed on this infrastructure. call a configuration. In contrast to purely analytical models, the state of network and applications can change over time through the use of events, as illustrated in Figure 1. LEAF uses events only in two cases: To read and to update configurations. Read events can, for example, be emitted by an algorithm that performs power measurements at a certain time step to adapt its behavior. Examples of update events are changes to the current application placements or the infrastructure graph like adapting the latency of a network link or adding a new compute node. LEAF does not use events to simulate individual network packets. Every configuration contains all information necessary for a deeper analysis and can be inspected without any knowledge about future or past events. Modeling fog computing environments in this way enables easy-to-analyze experiments that can execute scenarios with thousands of devices and applications. Yet, our approach also has decisive advantages over purely analytical models: First, by allowing the model to change over time, heterogeneous environments with dynamically changing network topologies, mobility of edge devices, and varying workloads can be represented. Second, LEAF allows for online decision making, enabling research on dynamic energy-aware task placement strategies, scheduling algorithms, or traffic routing policies.
A. Infrastructure Model
As depicted in Figure 2, the network and computing infrastructure are represented as a weighted, directed multigraph I = (N, L), where N is the set of compute nodes. A compute node N i ∈ N may be resource-constrained, can have a location, and may be mobile. It can represent an entire cloud data center as well as mobile sensors with limited or no computing capacity. L is the set of network links between nodes. A network link L i ∈ L can represent individual wired or wireless connections as well as entire network routes such as WAN connections. Network links can be constrained by bandwidth and may have additional properties like latency that users can utilize for implementing routing policies. Edges are directed to allow modeling different constraints and power models for uplink and downlink. Multiple edges between nodes are allowed and can represent different available link types such as Wi-Fi and Bluetooth. The infrastructure graph can change during the simulation, as nodes are free to move around and can join or leave the network. Furthermore, any property, like the latency of a link, can change.
B. Application Model
LEAF considers all applications to be streaming applications and, hence, regards all communication between different application tasks as continuous data flows. Applications are represented by directed acyclic graphs A = (T, F ), where T is the set of tasks in the application, and F is the set of data flows between tasks. Tasks, as well as data flows, have certain resource requirements. Applications can be placed on the fog by mapping their tasks to compute nodes, and their data flows to network links. Application placements are described in more detail below. A task T i ∈ T demands certain resources from its compute node. A compute node can host as many tasks as its resources allow. There exist three different kinds of tasks that are responsible for emitting, processing, and retrieving data: 1) Source tasks are bound to a specific compute node, for example, a sensor-equipped or mobile device. They are constantly emitting data and have at least one outgoing edge and no incoming edges. 2) Processing tasks can be freely placed on any compute node N i ∈ N that fulfills their resource requirements.
Processing tasks have at least one incoming edge and at least one outgoing edge. 3) Sink tasks are again bound to a specific compute node and have at least one incoming edge and no outgoing edges. Examples of data sinks are cloud storages or enduser terminals.
A data flow F i ∈ F represents a continuous stream of data between two tasks, stated in bits/s. Like any parameter, the data rate can change during the simulation. A network link can "host" as many data streams as its bandwidth allows. Due to its abstract network model, LEAF is not suitable to explicitly model complex effects resulting from network congestion like queuing delay or packet drops.
When placing an application, its tasks have to be mapped onto compute nodes, which is illustrated in orange in Figure 2. Since the assignment of source and sink tasks is fixed, task placement strategies have to determine the placement of processing tasks. After the placement, a user-defined routing policy searches for an optimal path in the network between every pair of connected tasks. If there is no node that meets the resource requirements or if no sufficient path can be found, the placement failed.
Typically, processing tasks aggregate and filter the data stream in a way that outgoing data flows require less bandwidth than incoming data flows. Consequently, the placement of processing tasks close to the source tasks generally reduces network usage and, thus, energy consumption. Finding optimal placements in dynamic, resource-constrained, distributed, and heterogeneous environments is a complicated problem and a focus of research [17]. LEAF enables application placement strategies that take the infrastructure's energy consumption into account.
IV. POWER MODELS
We model infrastructure power usage by assigning each compute node and network link its own power model. This enables users to determine the power usage of data centers, edge devices, and network links individually. A power model P (t) is a function that returns the current power usage of its corresponding entity at any point in time t during the simulation. Power usage is reported as the sum P static +P dyn , where P static is the static (load-independent) power usage and P dyn the dynamic (load-dependent) fraction. Power models may maintain a state which can be used to implement energysaving mechanisms (see Section IV-D).
To create load profiles of infrastructure components, their power models can be called through periodic events. Additionally, other simulated components can call power models at runtime to adapt their behavior or update their state. Examples of such components are energy-aware task placement strategies or hardware like batteries that update the state of charge.
A. Linear and Non-Linear Power Models
Research shows that the power usage of infrastructure can often be modeled with sufficient accuracy as a linear function that is only based on the entity's current load C(t) [18], [19]. Linear power models are beneficial since they are computationally efficient and easy to comprehend. They are described as where C(t) σ represents P dyn . The variable σ determines the incremental energy per load. For example, for network equipment, σ represents the energy consumed per bit of transferred data (J/bit). When modeling resource-constrained compute nodes or network links that have a maximum load C max with power usage P max , σ can be expressed as: An example for more complex entities, where a simple, linear function may not model the power usage well, are compute nodes representing entire data centers. Data centers incorporate several different hosts and require additional power for operational overhead like cooling and lighting. An exemplary approach for modeling this kind of compute node is to define a power model P Hi for each host H i and to aggregate their results to P dc . To account for operational overhead, we can multiply the result with the data center's power usage effectiveness (PUE): Compute nodes and network links can also apply nonlinear power models and power models with multiple inputs to improve the accuracy of the simulation. An example is the power model for wireless network links of [20], which, in addition to the load, also takes the distance of communicating devices into account. However, more complex power models will lead to more computationally intensive simulations, hence longer runtimes.
B. Power Models for Shared Resources
When describing the power consumption of shared resources such as cloud data centers, mobile base stations, or entire WAN links, it is not meaningful to specify static power usage. For example, it is impossible to know at which point an externally operated data center will need to switch on or off a new physical machine. Many of these resources are assumed to scale on demand, hence defining a P max is not even possible. For these reasons, P static = 0 for power models of shared infrastructure. Users must estimate a meaningful σ that directly incorporates a fraction of the static consumption.
By modeling shared resources without static power usage, we implicitly assume this infrastructure to be energyproportional. Nevertheless, there are ways to incorporate inefficiencies into the parameters. In practice, servers as well as routers are rarely used to full capacity but are over-provisioned to ensure performance and availability. Equivalently to the energy model proposed by [4], users can estimate and incorporate an operational capacity fraction U into a power model. The effect of U is illustrated in the staircase curve depicted in Figure 3.
C. Power Models for Network Links
Although we assign power models directly to edges in the infrastructure graph, in reality, energy is not consumed by the cable or wireless connection itself but by networking equipment such as routers or wireless transmitters. Network link power models hence represent the aggregated power usage of all networking equipment involved to transfer data from one compute node to another, and σ describes the incremental energy per bit (J/bit). Static power usage should be attributed to the adjacent compute nodes, to assure it correctly drops to zero when the node is shut off. Intermediate routing devices are considered shared infrastructure and their consumption should be incorporated directly into the network link's σ. Table I depicts an exemplary parameterization of a WAN link between a mobile device and a cloud server.
For wireless connections, power usage characteristics of uplink and downlink usually differ [21], [22]. Since infrastructure is represented by a directed multigraph in LEAF, it allows modeling asymmetrical power usage characteristics by assigning different power models to the respective directions.
D. Energy-saving Mechanisms
Energy-saving mechanisms aim to reduce static power usage of hardware during periods of low utilization by throttling or powering off parts of the system. They can significantly influence overall energy consumption by pushing infrastructure towards operating energy-proportionally. Such load profiles can be modeled in LEAF by making P static , which usually describes the load-independent power usage, indirectly loaddependent. For example, a common strategy in data centers is to consolidate workload on a few hosts and to power off the remaining hosts. P static of such a data center could be modeled as a step function, similar to the example in Figure 3.
Furthermore, to evaluate the impact of even more complex energy-saving mechanisms, users can make use of the fact that power models can maintain state. For example, [23] presented a precise model for dynamic voltage and frequency scaling (DVFS), which requires the current and past utilization of the CPU to implement different strategies. LEAF's power models 2 http://tools.cisco.com/cpc, accessed 2020- [10][11][12][13][14][15][16][17] can store past utilization states of their assigned entity and take them into account when computing the current power usage.
E. Tracing Back Power Usage to Applications
To support the implementation of energy-conserving application placement strategies, LEAF allows tracing back the infrastructure's power usage to the responsible applications. The power consumption of an application is defined as the sum of all power usage that its tasks and data flows cause on their allocated resources, see Figure 2. Depending on the scenario users may decide to only attribute dynamic power usage, or dynamic and static power usage. For example, if the underlying resource is powered on, regardless of its utilization, the static energy consumption does not have to be attributed to any applications since they have no influence on it. However, if the resource can be powered off when it is idle, the placed resources actually cause the energy usage. In this case, the static energy should be attributed to all tasks that currently run on the node depending on their fraction of the total load.
V. EVALUATION
We created two open source implementations of LEAF in Java and Python. The respective GitHub repositories are referenced in Section I. The following evaluation uses the Java implementation, based on the CloudSim Plus [24] simulator, and simulates different fog architectures and application placement strategies in a smart city traffic scenario. Figure 4 shows the average number and speed of taxis generated per minute. The infrastructure graph consists of four kinds of compute nodes: The cloud, fog nodes, STLs, and taxis. Taxis are connected via Wi-Fi, e.g. IEEE 802.11p, to nearby STLs. The mapping of data flows to network edges is updated periodically as taxis move around. STLs communicate with other close-by STLs via Wi-Fi too, effectively forming a mesh network. Additionally, they are equipped with 4G LTE modules connecting them to the cloud node via WAN. Table II shows the infrastructure parameterization. Only cloud and fog nodes have computing capacities stated in millions of instructions per second (MIPS). Since the load on STL and taxi nodes is the same in all experiments, their power usage is not modeled in this evaluation. All other compute nodes and network links have linear power models. The cloud is considered shared infrastructure and has no static but high dynamic power usage. Energy per bit for WAN links was configured as the example shown in Table I. Direct Wi-Fi communication between STLs consumes less energy than communication with taxis because they always have a direct line of sight and deploy energy-efficient, highthroughput access points.
A. Experimental Setup
We simulate a smart traffic system comprising two kinds of applications: CCTV applications process data recorded by cameras deployed at STLs. The source task, located at the STL, sends 10 Mbit/s of video data to a processing task that requires 30 000 MIPS and is responsible for traffic monitoring, enforcing traffic laws and automatic incident detection. The 200 kbit/s of resulting data are sent to the sink task located in the cloud for further analysis and storage. 16 of these applications are running in our scenario, one for each STL. V2I applications are vehicle-to-infrastructure, smart traffic management applications that control traffic lights to ensure maximum throughput of public transport. Each taxi on the map streams 100 kbit/s of sensor data to a processing task which requires 7000 MIPS and forwards 50 kbit/s to all STLs on the way of the taxi. The number of applications running depends on the number of taxis on the map.
B. Experiments
We conducted eight different experiments to demonstrate that our model enables research on energy-efficient fog architectures, task placement strategies and energy-saving mechanisms. Figure 5 provides
1) Cloud Only:
In the first experiment, all processing tasks of both application types were placed in the cloud. There exist no fog nodes. Figures 6a and 6d depict the consumption of infrastructure components and application types over time. We can observe that the major part of the power consumption, namely 66.4 %, can be attributed to the WAN as all raw sensor data have to travel to the processing tasks placed in the cloud. The accumulated power required by V2I applications correlates with the number of taxis on the map.
2) Fog Only: In experiment Fog 6, six traffic light systems were equipped with fog nodes, to avoid data transfer over the power-intensive WAN. These nodes provide sufficient computing capacity so no tasks have to be offloaded to the cloud. Processing tasks are distributed evenly across the fog nodes. As expected, the power usage of network decreased sharply. The fog nodes themselves are now the most relevant power consumers, accounting for 94.7 % of the total energy consumption. A major fraction of this, namely 57.8 %, is static power consumption: The six fog nodes require 100 W of static power each, totaling in 14.4 kW during a day.
Although the computational and network load required by the CCTV applications is constant during the entire simulation, the reported power consumption varies over time. This is because we allocate the static power consumption of fog nodes proportionally to applications running on them and fog nodes are utilized inefficiently in this experiment. Especially at night when only a few taxis are on the road, the relative power demanded by the CCTV applications rises.
3) Fog and Cloud: Experiments Fog 1 to Fog 5 contain one to five fog nodes, respectively. Tasks are still distributed evenly across the available fog nodes, but once all fog nodes are running at more than 85 % capacity, tasks are being offloaded to the cloud. The lowest overall energy consumption was achieved in experiment Fog 4, saving 2300 Wh compared to Fog 6. Four fog nodes provide enough capacity to host all CCTV processing tasks and around 125 V2I processing tasks. Although during some periods of the day more than 125 taxis are on the map simultaneously, the savings on static fog node power usage outweigh the additional cloud and WAN power usage caused by task offloading with four fog nodes. 4) Energy-Saving Mechanism: Experiment Fog 6s extends experiment Fog 6 with an adaptive task placement strategy and energy-saving mechanism. This time, if a fog node is idle for five seconds, it will be put to sleep, reducing its static power consumption of zero. In order to fully exploit this energy- saving mechanism, the task placement algorithm tries to consolidate as much work as possible on a minimum number of nodes to maximize the number of idle fog nodes. Individual fog nodes are still only utilized up to 85 % capacity. Figure 6c shows that the overall static power usage of fog nodes is now no longer constant. All six fog nodes are only active for less than 2 hours during the day, while during the night only two out of six are active. Consequently, the power usage of applications was reduced too, see Figure 6f. Resulting from the improved placement strategy, CCTV applications are now responsible for less static power usage, and, thus, have a very stable load profile. V2I applications benefit from the energysaving mechanism by becoming more power proportional. At night, the attributed power usage of all V2I applications drops below 80 W. Figure 7 displays how experiment Fog 6s undercuts the second best performing experiment Fog 4 at different times of the day. When there is little traffic in the city, Fog 6s benefits from highly reduced static power usage of shut-off fog nodes. In times of high utilization, Fog 6s continues to provide sufficient resources to process all data in the fog layer, avoiding expensive WAN traffic for offloading. At times of average utilization, the load profile of both experiments is almost identical.
C. Analysis
So far the evaluation has shown that we have successfully met our Requirements (1), (2), and (3): LEAF was used to model a realistic fog computing scenario with different kinds of compute nodes, network links and applications. By assigning individual power models to different parts of the infrastructure, we were able to analyze the energy footprint of different fog node deployments in order to find the most energy-conserving configuration. In the last experiment, we demonstrated how our model enables research on dynamic task placement strategies and energy-saving mechanisms.
To evaluate Requirement (4), Performance and Scalability, we analyzed the runtimes of simulations. Each presented experiment simulates around 46 500 taxis executing around 330 000 tasks and finishes in less than 50 seconds on a single core of a 1.4 GHz Intel Core i5. A series of further experiments show that in the outlined scenario, the only algorithmic component that shows non-linear growth is the algorithm for finding shortest paths between tasks. Even when scaling experiments by factor 10, namely 465 000 taxis and 3 300 000 tasks, they had a median runtime of 613 seconds. Consequently, the presented model is suitable to simulate large-scale experiments several magnitudes faster than real time on commodity hardware.
VI. CONCLUSION
This paper presents LEAF, a simulator for modeling large energy-aware fog computing environments. LEAF features a holistic but granular energy consumption model that covers data centers, edge devices, and network as well as applications which are running on this infrastructure. By combining analytical and discrete-event modeling, the proposed model enables the simulation of thousands of streaming applications on a distributed, heterogeneous, and dynamic infrastructure. The publicly available implementation of LEAF was evaluated within a realistic smart city traffic scenario and proved to be a valuable tool for research on energy-conserving fog computing architectures, task placement strategies, and energy-saving mechanisms.
In the future, we plan to extend our model to provide timebased and location-based calculations of the carbon emissions and electricity costs, thus enabling research on infrastructures and algorithms optimized for a low environmental footprint and operating expenditure. Moreover, we plan to integrate LEAF with relevant simulators for network, urban traffic, or electric grids to co-simulate and evaluate more realistic scenarios. | 2021-03-03T02:15:44.679Z | 2021-03-01T00:00:00.000 | {
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257312688 | pes2o/s2orc | v3-fos-license | A new class A beta-lactamase gene blaCAE-1 coexists with blaAFM-1 in a novel untypable plasmid in Comamonas aquatica
Antimicrobial resistance, especially carbapenem resistance, poses a serious threat to global public health. Here, a carbapenem-resistant Comamonas aquatica isolate SCLZS63 was recovered from hospital sewage. Whole-genome sequencing showed that SCLZS63 has a 4,048,791-bp circular chromosome and three plasmids. The carbapenemase gene blaAFM-1 is located on the 143,067-bp untypable plasmid p1_SCLZS63, which is a novel type of plasmid with two multidrug-resistant (MDR) regions. Notably, a novel class A serine β-lactamase gene, blaCAE-1, coexists with blaAFM-1 in the mosaic MDR2 region. Cloning assay showed that CAE-1 confers resistance to ampicillin, piperacillin, cefazolin, cefuroxime, and ceftriaxone, and elevates the MIC of ampicillin-sulbactam two-fold in Escherichia coli DH5α, suggesting that CAE-1 functions as a broad-spectrum β-lactamase. Amino acid sequences analysis suggested that blaCAE-1 may originate from Comamonadaceae. The blaAFM-1 in p1_SCLZS63 is located in a conserved structure of ISCR29-ΔgroL-blaAFM-1-ble-ΔtrpF-ΔISCR27-msrB-msrA-yfcG-corA. Comprehensive analysis of the blaAFM-bearing sequences revealed important roles of ISCR29 and ΔISCR27 in the mobilization and truncation of the core module of blaAFM alleles, respectively. The diverse passenger contents of class 1 integrons flanking the blaAFM core module make the complexity of genetic contexts for blaAFM. In conclusion, this study reveals that Comamonas may act as an important reservoir for antibiotics-resistance genes and plasmids in the environment. Continuous monitoring for the environmental emergence of antimicrobial-resistant bacteria is needed to control the spread of antimicrobial resistance.
Scientific Reports
| (2023) 13:3634 | https://doi.org/10.1038/s41598-023-28312-w www.nature.com/scientificreports/ demonstrated that hospital sewage effluent creates a niche where pathogens acquire novel antibiotic resistance genes (ARGs), including carbapenemases genes. Hem et al. 16 isolated a subset of carbapenem-resistant Comamonas strains from wastewater, of which most possess novel unknown resistance mechanisms for carbapenems. Therefore, wastewater is an ideal place to identify novel ARGs. To date, carbapenem resistance genes bla NDM 17 , bla IMP- 8 18 , and bla GES-5 16 have been reported in Comamonas isolates. AFM-1 is a newly emergent carbapenemase that was first identified in a clinical Alcaligenes faecalis strain AN-70 in China and was later reported in Pseudomonas aeruginosa 19 and Aeromonas hydrophila 20 , in which ISCR elements are involved in the mobilization of bla AFM-1 . In silico analysis with the GenBank database indicated four variants of AFM (AFM-1 to -4) that were also present in Stenotrophomonas maltophilia, Bordetella trematum, as well as Comamonas isolates. In this study, we described a novel type of multidrug-resistance plasmid carrying the carbapenemase gene bla AFM-1 in a Comamonas aquatica strain from hospital sewage. Of note, we also characterized a novel class A serine β-lactamase gene, bla CAE-1 , which coexists with bla AFM-1 on the plasmid. In addition, we performed a comprehensive genomic comparison of bla AFM -bearing sequences to gain a better understanding of the dissemination patterns of this novel carbapenemase gene.
Materials and methods
Bacterial isolation and in vitro susceptibility testing. C. aquatica SCLZS63 was recovered from the sewage outlet of the affiliated hospital of Southwest Medical University, Sichuan Province, China, in November 2019. As previously described 21 , 5 ml of water sample was concentrated by centrifugation for 5 min at 5000 g, and the sediment was resuspended in sterile 0.9% NaCl solution, then, the bacterial suspension was plated onto MacConkey agar containing meropenem (2 μg/ml) and incubated for 24 h at 37 °C. A single colony was picked, and initial species identification was performed by sequence analysis of 16S rRNA gene after PCR amplifying and Sanger sequencing 22 . The susceptibility to ceftazidime, cefotaxime, aztreonam, meropenem, and imipenem for SCLZS63 was examined by using the broth microdilution method and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for other non-enterobacterales bacteria 23 .
Genome sequencing and analysis. Genomic DNA of SCLZS63 was obtained using the QIAamp DNA Mini Kit (Qiagen), and the purified DNA was subjected to whole-genome sequencing on a HiSeq 2000 platform (Illumina, San Diego, CA, USA) using a paired-end library with an insert size of 150 bp, followed by the long-read MinION Sequencer (Nanopore, Oxford, UK). The de novo hybrid assembly of the Illumina reads and MinION reads were carried out by using Unicycler v0.4.3 24 . Gene annotation for the assembled genomes was performed with the RAST tools 25 and BLASTp/BLASTn searches against the UniProtKB/SwissProt database 26 . Bacterial precise species were identified using pairwise ANI analysis between strain SCLZS63 and reference genomes of Comamonas with the online software JSpeciesWS (https:// jspec ies. riboh ost. com/ jspec iesws/). A > 96% ANI cut-off was used for species circumscription 27 . Plasmid incompatibility types, antibiotic resistance genes, and insertion elements were predicted using PlasmidFinder 2.1 (95%, minimum threshold for identity; 60%, minimum coverage) 28 , ResFinder (90%, minimum threshold for identity; 60%, minimum coverage) 29 , and ISfinder 30 .
Phylogenetic analysis. Amino acid sequences of 17 class A β-lactamases were retrieved from the Beta-Lactamase DataBase (BLDB, http:// bldb. eu/), and were utilized for the phylogenetic analysis. Sequences were aligned using the program Clustal W 31 . A maximum-likelihood tree was generated by MEGA 6 software 32 , with 1000 bootstrap replicates, and was then annotated using iTOL 33 . Gene cloning. PCR amplification of the complete coding sequence of bla CAE-1 and its promoter region from C. aquatica SCLZS63 was performed using the primers bla CAE -F: 5′-cagcaaatgggtcgcggatccGCT TAC TTT CAC TCA TGA CGT CAC C-3′and bla CAE -R: 5′-gtggtggtggtggtgctcgagGGA TGT TGG AAG ACC CGA CC-3′. The resulting PCR fragment was then ligated into an expression vector pET28a using a ClonExpress® II One Step Cloning Kit (Vazyme, China) to construct pET28a-bla CAE-1 , which was introduced into Escherichia coli DH5α by chemical transformation. Potential transformants containing the recombinant plasmid were selected on Luria-Bertani (LB) agar plates containing 50 mg/L kanamycin and verified by PCR assays. The susceptibility to antimicrobial agents (ampicillin, piperacillin/tazobactam, ampicillin/sulbactam, cefazolin, ceftriaxone, ceftazidime, cefotaxime, cefepime, aztreonam, ertapenem, meropenem, and imipenem) for the recombination strain was performed using the broth microdilution method according to the CLSI guidelines with E. coli strain ATCC 25922 as the quality control strain. E. coli DH5α containing the empty pET28a served as a negative control.
Conjugation and electroporation experiments.
Conjugation experiments were performed using both broth-and filter-based methods, with the azide-resistant E. coli J53 as the recipient, as described previously 34 . Equal amounts of donor and recipient cells at the exponential stage (the optical density at 600 nm reaches ~ 0.5) were mixed and incubated at 37 °C in LB broth or on the filter that was placed on an LB agar plate overnight. Subsequently, cells were resuspended and diluted in 0.9% NaCl, and potential transconjugants were selected on LB agar plates containing 150 µg/ml sodium azide and 4 µg/ml cefotaxime. Conjugation assays were repeated with different donor/recipient ratios.
Electroporation was carried out with E. coli DH5α as the recipient. Plasmids of C. aquatica SCLZS63 were extracted using the E.Z.N.A. plasmid Mini Kit I (OMEGA, Bio-Tek, USA), verified by agarose gel electrophoresis, and then transferred by electroporation (Micro-Pulser electroporator; Bio-Rad, USA) into DH5α competent cells. Transformants were selected on LB agar plates containing 4 µg/ml cefotaxime. The presence of bla CAE-1 in the transformant was examined by PCR assays with primers bla CAE -F/R. www.nature.com/scientificreports/ Comparative analysis of bla AFM -bearing sequences. To obtain the bla AFM -bearing sequences, a BLASTn with standard options was performed with the nucleotide sequences of bla AFM-1 (GenBank accession no. NG_063835) as a query in the NCBI GenBank database. Plasmids and chromosomes with a full-length hit to bla AFM-1 (100% query coverage and ≥ 99.88% identity) were selected. Alignments of the bla AFM -bearing sequence were performed using BLASTn and visualized with Easyfig v 2.2.3.
To determine whether CAE-1 mediates resistance to β-lactams or not, bla CAE-1 was cloned into the pET28a vector and transformed into E. coli DH5α. Compared with the control strain DH5α carrying the empty pET28a, the recombinant strain DH5α/pET28a-bla CAE-1 exhibited resistance to some β-lactams tested, including ampicillin, piperacillin, cefazolin, cefuroxime, and ceftriaxone (Table 2), and the MIC for ampicillin-sulbactam increased for two-fold. The acquisition of bla CAE-1 had no effect on the MICs of carbapenems and aztreonam. This finding implies that CAE-1 has broad-spectrum activity profiles, and functions as broad-spectrum β-lactamase.
The mobilization and possible origin of bla CAE-1 . We screened the presence of bla CAE-1 in GenBank database by BLASTn (Accessed by 20 August 2022), and four matches were found, including one plasmid from C. aquatica (CP079746, China, water, 2019), and three chromosomes of P. aeruginosa (CP042967, patient, Thailand, 2016), C. aquatica (LR813086, Spain, Water, 2020) and Comamonas thiooxydans (CP063057, China, patient, 2019). Analysis of the adjacent genetic elements of bla CAE-1 in p1_SCLZS63 and the above four bla-CAE-1 -carrying sequences showed that bla CAE-1 is always reversely located at the immediate upstream of the LysR family transcriptional activator-encoding gene ampR (Fig. 2), which consists with the commonly seen lysRaccompanied class A β-lactamases as described previously 36 . In all sequences except pB1A, the bla CAE-1 -ampR element is bounded by an intact IScaq2 (upstream of bla CAE-1 ) and a ΔIScaq2 (immediately downstream of bla-CAE-1 ) that is truncated by a parallel IScaq1 (Fig. 2). IScaq1/IScaq2 (Accession no. LR813086/CP063057) are IS5 family transposases that are initially identified in C. aquatica in the GenBank database. However, the flanking IScaq2 elements are in opposite orientations, which denies the assumption of an IScaq2-based composite transposon. In pB1A, the bla CAE-1 -ampR element is bracketed by two IS110 family transposases, which are in the reversed orientation as well (Fig. 2). The exact mechanism of the mobilization of bla CAE-1 remains unclear.
To investigate the possible origin of bla CAE-1 , BLASTp search of CAE-1 against non-redundant protein database was performed. We found that CAE-1 shows significant amino acid identity with chromosomally encoded class A β-lactamase of bacteria mainly from Comamonas and Acidovorax sp., which both belong to the family Comamonadaceae. Additionally, we found that the average GC-content for the immediate genetic background of the bla CAE-1 gene (namely, the bla CAE-1 -ampR element) is 63.81%, which is close to the chromosomal GC-content of Comamonas (64.53% for SCLZS63). These findings suggest that Comamonadaceae may be an ancestral source of the bla CAE-1 gene. www.nature.com/scientificreports/ includes regions responsible for plasmid replication (repA), maintenance (parAB, umuCD), and conjugative transfer (tra genes), and it could not be assigned into any known incompatibility group. No similar plasmids were found in the GenBank database using the backbone sequences of p1_SCLZS63 as a query, suggesting that p1_SCLZS63 is a novel type of plasmid. Within the backbone, p1_SCLZS63 harbors two multidrug-resistant (MDR) regions and an arsenate resistance operon (arsCDABCH) (Fig. 3A). In the 7.4-kb MDR1 region, the class 1 integron intI1-dfrA5-aac(6')-IIa-qacEΔ1-sul1 is bounded by an IS5 element (upstream) and an IS6100 (downstream). The 32-kb MDR2 region shows a complex structure and is a mosaic with areas of diverse origin (Fig. 3B). The Tn3-borne defective mercury resistance operon (mer) and the neighboring bla CAE-1 region were remarkably similar (99.9% identity, 100% coverage) to a region on the chromosome of P. aeruginosa strain PA99 (Accession no. CP042967). The msr(E)-mph(E) unit flanked by IS26 is universally found in many bacterial families, such as Enterobacteriaceae and Moraxellaceae. The bla AFM-1 region resembled that on the plasmid pSS332-218k (Accession no. CP071152) from a clinical Aeromonas caviae in Zhejiang, China. In this region, two copies of intI1 (one complete and one truncated)-sul1 element in parallel orientation bracketed the core bla AFM-1 platform, wherein Ceftriaxone www.nature.com/scientificreports/ the bla AFM-1 is located at a conserved fragment ISCR29-ΔgroL-bla AFM-1 -ble-ΔtrpF-ΔISCR27-msrB-msrA-yfcG-corA, as had been reported previously in other plasmids 19,20 .
To determine the transfer ability of p1_SCLZS63, conjugation experiments were carried out with E. coli J53 as the recipient. Despite repeated attempts, no transconjugants containing p1_SCLZS63 were obtained. In addition, the transfer of p1_SCLZS63 into E. coli DH5α by electroporation was also unsuccessful after several attempts.
Genetic contexts of bla AFM alleles. A total of 14 bla AFM -bearing plasmids (n = 11) and chromosomes (n = 2) were retrieved from the GenBank database (Accessed on 19 October 2022). bla AFM was identified on the chromosomes of S. maltophilia, B. trematum, and P. aeruginosa, and it was also carried by different types of plasmids with various sizes (61,915-495,621 bp) from Comamonas testosterone, A. caviae, P. aeruginosa, Pseudomonas asiatica, A. faecalis, and C. aquatica from China. These bla AFM -bearing plasmids are generally untypable, except that some IncP-2 type plasmids (pAR19640, pNDTH9845, and pWTJH17) carry bla AFM-2 in P. aeruginosa, and an IncW plasmid pAN70-1 carries bla AFM-1 in A. faecalis.
Generally, two kinds of bla AFM -bearing core modules were identified, namely ISCR29-ΔgroL-ΔfloR-bla AFM -ble-ΔtrpF-ΔISCR27-msrB-msrA-yfcG-corA (designated type A) and its truncated version www.nature.com/scientificreports/ ISCR29-ΔgroL-ΔfloR-bla AFM -ble-ΔtrpF-ΔISCR27 (type B). The truncation of type B seems to have resulted from the recombination event of ΔISCR27 (Fig. 4). Almost all the bla AFM-1 and bla AFM-4 genes are found in the type A module, except that bla AFM-1 in pMD9A is in the type B module, in which form bla AFM-2 and bla AFM-3 genes are embedded, and that the bla AFM-1 -bearing type A module in pAN70-1 is disrupted by a Tn6346-like transposon 19 .
In addition, we found that almost all the bla AFM -bearing core modules are always flanked by class 1 integrons (Fig. 4). The intI1-sul1-bla AFM-1 core module-ΔintI1-sul1 in p1_SCLZS63 in this study seems to be an ancestor structure, from which class 1 integrons with different cassette arrays surrounding the bla AFM core module have arisen.
Discussion
Antimicrobial resistance represents a growing threat to medical care. Infections caused by carbapenem-resistant bacteria are usually associated with poor prognoses and increased morbidity and mortality rates 40 . Close monitoring of carbapenem-resistant bacteria in the hospital sewage is essential. Comamonas, especially, carbapenemresistant Comamonas, are abundant in wastewater 16 , while their genome characteristics of antibiotic resistance are poorly characterized. In this work, we isolated a carbapenem-resistant C. aquatica from hospital sewage, and its genetic information of drug resistance was characterized by high-resolution WGS. C. aquatica has been isolated as the causative agent of bacteremia and septic shock 41 . The prevalence of multidrug-resistance, especially carbapenem resistance in C. aquatica warrants further public health surveillance. Ambler classe A β-lactamases are prevalent and diverse considering other molecular classes, and they represent the most important enzymatic source of both natural and acquired resistance to β-lactams in Gram-negative bacilli 38 . In this study, a new enzyme CAE-1 has been added to the list of class A serine β-lactamase. It exhibits resistance to ampicillin, piperacillin, cefazolin, cefuroxime, and ceftriaxone, as revealed by the in vitro susceptibility analysis. To confirm this, kinetics analysis on pure CAE-1 enzyme would have been informative. With this goal in mind, the bla CAE-1 gene without its promoter region was cloned in a pET28a expression vector and introduced in an E. coli expression strain. However, repeated attempts at expressing and purifying a N-terminus his-tagged version of the CAE-1 enzyme (expected molecular mass of ~ 34 kDa) proved unsuccessful. Possible reasons for why the heterologous expression did not work include low expression under detection limit and an inappropriate host. Further work will be needed to understand the enzyme kinetics of CAE-1.
Plasmids play a vital role in the dissemination of ARGs via horizontal gene transfer. Antibiotic resistance plasmids are rarely reported in Comamonas. Two IncP-1 plasmids were previously reported in Comamonas from aquatic environments, one of which was associated with the degradation of dyes 42 , and the other one was involved in resistance to heavy metal and oxidative stress 16 . None of the two IncP-1 plasmids carry any ARGs. In the strain SCLZS63, most ARGs are located on the untypable plasmid p1_SCLZS63, including the carbapenem resistance gene bla AFM-1 . The previously reported carbapenemase genes bla GES-5 , and bla IMP-8 in Comamonas are both chromosomally located 16,18 . The emergence of the carbapenemase gene on the plasmid in Comamonas has important public health implications, which demands more attention. The conjugation experiments indicated that the bla AFM-1 -harboring p1_SCLZS63 is non-transmissible in this case. While the genomic sequences presented here infer the transfer potential of p1_SCLZS63, the unsuccessful conjugation might result from the exceptionally low transferability that is below detectable limits, or the non-replication of this plasmid due to the unsuitability of E. coli J53 recipient strain used in this study for p1_SCLZS63 from C. aquatica. Electroporation of p1_SCLZS63 into E. coli DH5α was also not successful, which again implies that p1_SCLZS63 may not be readily maintained in a different host of E. coli cells.
AFM is a newly identified subclass B1b metallo-β-lactamase, which shows partial identity to the widespread NDM 19 . bla AFM has been found in different species of non-fermenting Gram-negative bacteria and is spreading in clinical P. aeruginosa isolates at a low rate in China 43,44 . ISCR elements are known to move and pick up adjacent genetic components via a rolling-circle mechanism 45 . In the cases of bla AFM alleles, ISCR29 may have initially acquired the ΔgroL-bla AFM -ble-ΔtrpF-ΔISCR27-msrBA-yfcG-corA-ΔISCR27 region and mobilized it upstream of an intI1-sul1 genetic segment, meanwhile truncating the intI1 gene, followed by a second insertion downstream of the second intI1-sul1 segment. The intI1-sul1-bla AFM-1 core module-ΔintI1-sul1 structure in plasmid like p1_SCLZS63 serves as the intermediate source for the dissemination of bla AFM , most likely by recombination events. The flanking class 1 integrons have acquired a variety of passenger genes in the subsequent genetic actions, generating complex genetic contexts for bla AFM .
Conclusions
In the present study, we identified and characterized a novel class A β-lactamase gene bla CAE-1 conferring resistance to broad-spectrum cephalosporin, from an environmental C. aquatica isolate, where bla CAE-1 coexists with the carbapenemase gene bla AFM-1 in a mosaic MDR region on a novel type of plasmid. This finding highlights the importance of the environment as a reservoir of novel antibiotic resistance plasmids and resistance determinants. Effective surveillance is required for understanding the transmission and ongoing evolution of this multidrugresistance plasmid in clinical settings.
Data availability
The complete sequences of the C. aquatica SCLZS63 chromosome and plasmids p1_SCLZS63, p2_SCLZS63, and p3_SCLZS63 have been submitted to GenBank with accession numbers CP104279 to CP104282, respectively. | 2023-03-04T14:33:16.685Z | 2023-03-03T00:00:00.000 | {
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187618494 | pes2o/s2orc | v3-fos-license | A Comparative Study of Effectiveness of Honey and Povidone Lodine in Healing of Surgical Wound
Introduction: Honey is a viscous, supersaturated solution derived from nectar modified by the honeybee. Honey has been used as a remedy in wound care and evidences have suggested accelerated wound healing. The aim and objective of the study was to compare the efficacy of honey and povidone iodine in different types of wound management Material and Methods: A longitudinal prospective study done in R.G. Kar Medical college and Hospital on 100 patients included following inclusion and exclusion criteria and were equally divided in two groups; one received honey dressing and other povidone-iodine. Clinical examination with history, pain scored by visual analog scale (VAS) and wound surface area were noted. Outcome was analysed at 10 days, 1 month and 3 months following intervention. Result: Mean age group in honey dressing group was 43.8 years and in povidone-iodine group was 43.91 years. Most of them had diabetic ulcers. Outcome in either group was comparable in terms of the etiology (p= 0.993). Ulcers at different locations had insignificant effect on outcome in either group (p= 0.836). Thus, etiology and location has no significant effect on the dressing material. Diabetes, smoking and alcoholism also had no significant effect in either group (p= 0.841, 0.067, 0.235 respectively). VAS score for pain was more reduced from baseline in honey dressing group than povidone-iodine group after both 1 and 3 months of intervention which was significant. Surface area of wound was also reduced significantly more in honey dressing group than povidone-iodine group after 1 and 3 months. However, after 10 days of intervention the outcome was comparable and insignificant. Conclusion: Honey appears to be more effective in wound healing than povidone-iodine. Key word: Honey, Povidone-iodine, Diabetic Ulcer, Pressure sore, Tegaderm Original research article Mukherjee, et al. Effectiveness of Honey and Povidone Lodine in Healing of Surgical Wound D143 International Journal of Contemporary Medical Research International Journal of Contemporary Medicine Surgery and Radiology Volume 3 | Issue 4 | October-December 2018 and it may cross 5.4% by the year 2025. According to Indian epidemiological data chronic wounds was reported as 4.5 per 1000 population whereas that of acute wounds was nearly doubled at 10.5 per 1000 population.2 In America 3–5% of all hospitalized patients have spinal cord injuries which suffer from ulcers. According to epidemiological data 225,000 spinal cord injury patients in the United States with about 9,000 new patients each year. Approximately 60% of diabetic patients develop pressure ulcers, and the range of annual cost estimate from $14,000 to $25,000 per patient for medical, surgical, and nursing care. The national expenditure cost of pressure ulcers is over $1.3 billion per year. Overall it is estimated that after 15 years the population will increase from 4 million to over 17 million individuals. Therefore, this health care problem is increasing at a dramatic rate.5 In America the cost of institutional care on the same is supposed to be US$ 1000 per day while no such estimates are available for Indian institutions, the same demographic study has projected market expenditure of over US$ 7 billion worldwide for provisions of wound healing properties. In India wound care is very expensive and especially with the diabetic population. The challenge was not only to improve wound care and treatment facilities but also stress on prevention among the population and heath care practitioner. The aim and objective of the study was to compare the efficacy of honey and povidone iodine in different types of wound management MATERIAL AND METHODS Present Longitudinal Non-randomised study was done in R.G Kar Medical College and Hospital, Kolkata on patients attending outdoor and emergency of R.G Kar medical college and hospital in the department of general surgery. Study period was from January 2016 to Oct 2017. It was done on 100 patients, 50 Patients in each group. Study parameters 1. Demographic Parameter: Age 2. Effect of Co-morbidities and habits on dressing technique: Diabetes, Smoking, Alcoholism 3. Outcome at different intervals after dressing: Pain Score by Visual Analog Scale, Surface area of wounds. Study method Inclusion criteria • 18 years old patients with chronic wound of duration >6 weeks • Patient presenting with infected wounds were initially treated with daily dressing-cleaning with normal saline and dressing with paraffine gauze along with surgical debridement and oral antibiotics based on bacterial wound culture report. Exclusion criteria: Patient with postoperative wounds, burn and skin graft donor sites, wound size> 5 cm in maximum diameter, known allergic to honey or povidone iodine. Study Technique: In this interventional prospective longitudinal non-randomised study, total 100 patients attending outdoor and emergency of department of surgery of R.G. Kar Medical College and Hospital during the study period was selected according to inclusion and exclusion criteria and divided into two groups with 50 subjects in each group. After written informed consent (Image 1: Patient Consent Form) has been taken and obtaining approval from ethical committee (Image 2: Ethical Approval Form), one group was dressed with honey and the other with povidone iodine. The baseline pain score by VAS and Surface area of the wound and comorbidities and habits were noted. After the intervention was done, the patients were followed up and pain score by VAS and surface area of wound was noted. The parameters as mentioned above were compared and analysed. STATISTICAL ANALYSIS Both descriptive and inferential statics will be used for tabulation, analysis and interpretation. Statistical measures like mean, SD and proportion as appropriate. In addition, we will consider using chi squared test of independence for categorical outcome variables. Study tools 1. Routine Blood Investigations: Complete hemogram, bleeding time, clotting time, fasting and postprandial blood sugar, serum Urea and Creatinine, 2. ECG, Chest X-ray 3. Limb X-ray and Doppler study where required 4. Visual Analogue scale for scoring pain
INTRODUCTION
Wounds are physical injuries that results in an opening and break of the skin that cause disturbance in the normal skin anatomy and function. They result in the loss of continuity of epithelium with or without the loss of underlying connective tissue. Wound may be produced by physical, chemical, thermal, microbial or immunological insult to the tissues. The process of wound healing consists of integrated cellular or biochemical events leading to the building of structural and functional integrity with regain of strength of injured tissues. Chronic wounds are a considerable burden to patients and the National Health Service (NHS). Current estimation indicate about 6 million people are suffering from chronic wounds worldwide. 1 The prevalence of chronic wounds in the community was reported as 4.5 per 1000 population, whereas an acute wound was about 10.5 per 1000 populations. 2 Some diseases like diabetes, immune-compromised conditions, ischemia and conditions like malnourishment, ageing, local infection, local tissue damage due to burn or gunshot often leads to delay in wound healing. Infection is the major complications of burn injury and is responsible for 50-75% of hospital deaths. 3 A lot of people are developing diabetes at a very younger age due to stressful life. It was reported that a lot of children having this chronic and fatal disorder. Long occurrence of these fatal disorders increases the chance of non-healing wounds. Chronic wounds are non-healing wounds will continue to rise with increasing the population ages, chronic diseases, and the poor nutrition available. Most chronic wounds are ulcers that are associated with ischemia, diabetes mellitus, venous stasis disease, or pressure. About 3 to 6 million people suffer with chronic wound in the United States in which persons are 65 years and older accounting for 85% of total cases. Non-healing wounds result in enormous health care expenditures, with the total cost estimated at more than $3 billion per year. 4 Diabetes mellitus is one of the most common factors for chronic wound due to metabolic disorders and 2.8% of the population suffers from this disease throughout the world
A B S T R A C T
Introduction: Honey is a viscous, supersaturated solution derived from nectar modified by the honeybee. Honey has been used as a remedy in wound care and evidences have suggested accelerated wound healing. The aim and objective of the study was to compare the efficacy of honey and povidone iodine in different types of wound management Material and Methods: A longitudinal prospective study done in R.G. Kar Medical college and Hospital on 100 patients included following inclusion and exclusion criteria and were equally divided in two groups; one received honey dressing and other povidone-iodine. Clinical examination with history, pain scored by visual analog scale (VAS) and wound surface area were noted. Outcome was analysed at 10 days, 1 month and 3 months following intervention. Result: Mean age group in honey dressing group was 43.8 years and in povidone-iodine group was 43.91 years. Most of them had diabetic ulcers. Outcome in either group was comparable in terms of the etiology (p= 0.993). Ulcers at different locations had insignificant effect on outcome in either group (p= 0.836). Thus, etiology and location has no significant effect on the dressing material. Diabetes, smoking and alcoholism also had no significant effect in either group (p= 0.841, 0.067, 0.235 respectively). VAS score for pain was more reduced from baseline in honey dressing group than povidone-iodine group after both 1 and 3 months of intervention which was significant. Surface area of wound was also reduced significantly more in honey dressing group than povidone-iodine group after 1 and 3 months. However, after 10 days of intervention the outcome was comparable and insignificant.
Conclusion: Honey appears to be more effective in wound healing than povidone-iodine. and it may cross 5.4% by the year 2025. According to Indian epidemiological data chronic wounds was reported as 4.5 per 1000 population whereas that of acute wounds was nearly doubled at 10.5 per 1000 population. 2 In America 3-5% of all hospitalized patients have spinal cord injuries which suffer from ulcers. According to epidemiological data 225,000 spinal cord injury patients in the United States with about 9,000 new patients each year. Approximately 60% of diabetic patients develop pressure ulcers, and the range of annual cost estimate from $14,000 to $25,000 per patient for medical, surgical, and nursing care. The national expenditure cost of pressure ulcers is over $1.3 billion per year. Overall it is estimated that after 15 years the population will increase from 4 million to over 17 million individuals. Therefore, this health care problem is increasing at a dramatic rate. 5 In America the cost of institutional care on the same is supposed to be US$ 1000 per day while no such estimates are available for Indian institutions, the same demographic study has projected market expenditure of over US$ 7 billion worldwide for provisions of wound healing properties. In India wound care is very expensive and especially with the diabetic population. The challenge was not only to improve wound care and treatment facilities but also stress on prevention among the population and heath care practitioner. The aim and objective of the study was to compare the efficacy of honey and povidone iodine in different types of wound management
Study method
Inclusion criteria • 18 years old patients with chronic wound of duration >6 weeks • Patient presenting with infected wounds were initially treated with daily dressing-cleaning with normal saline and dressing with paraffine gauze along with surgical debridement and oral antibiotics based on bacterial wound culture report.
Exclusion criteria: Patient with postoperative wounds, burn and skin graft donor sites, wound size> 5 cm in maximum diameter, known allergic to honey or povidone iodine.
Study Technique:
In this interventional prospective longitudinal non-randomised study, total 100 patients attending outdoor and emergency of department of surgery of R.G. Kar Medical College and Hospital during the study period was selected according to inclusion and exclusion criteria and divided into two groups with 50 subjects in each group. After written informed consent (Image 1: Patient Consent Form) has been taken and obtaining approval from ethical committee (Image 2: Ethical Approval Form), one group was dressed with honey and the other with povidone iodine. The baseline pain score by VAS and Surface area of the wound and comorbidities and habits were noted. After the intervention was done, the patients were followed up and pain score by VAS and surface area of wound was noted. The parameters as mentioned above were compared and analysed.
STATISTICAL ANALYSIS
Both descriptive and inferential statics will be used for tabulation, analysis and interpretation. Statistical measures like mean, SD and proportion as appropriate. In addition, we will consider using chi squared test of independence for categorical outcome variables.
Age group
Mean age was 43.8 (SD= 7.56) years in the honey group and 43.91 (SD = 6.02) years in the povidone iodine group.
Distribution of patients according to type of wound in the study groups
The study comprises of Diabetic foot ulcer, pressure bed sore, Traumatic ulcer and venous ulcer. All types of ulcers are comparatively equally divided in both groups. They are comparable as p value (0.993) is insignificant.
Distribution of patients according to location of ulcer
In our study we had ulcers at leg, ankle, foot, Dorsum, sole,
Distribution and Comparing study groups by p-values in terms of different co-morbidities and habits
In our study we considered and compared Diabetes, smoking and alcoholism in the two study groups. The results were insignificant since p value was >0.005. The p values were 0.841, 0.067, 0.235 for diabetes, smoking and alcoholism respectively in both groups.
Distribution according to outcome at the different time milestone
The outcome was observed in terms of the pain score assessed by Visual Analog Scale (VAS) and reduction of surface area (rSA) at 10 days, 1 month, 3 months after the study. The analysis showed insignificant changes at 10 days. The changes show significant difference in outcomes with honey dressing and povidone-iodine dressing at 1 month and 3 months after the study.
DISCUSSION
Majority of the subjects 50% in the honey dressing group and 52% in the povidone iodine dressing group-were having chronic wound due to diabetic foot. WHO estimated that 347 million people worldwide suffer from diabetes. 6 Diabetes currently affects more than 62 million Indians, which is more than 7.1% of the adult population. 7 The life time foot ulcer risk is 25% in diabetes. 9 We had used numerous strategies to treat wound infections, including topical and systemic administration of antibiotics and various antiseptic agents like Povidone-Iodine to kill bacteria or inhibit their growth. Decisions regarding choice of wound treatment involve two basic considerations: (1) how safe is the treatment and (2) how effective is the treatment. 9 Around 1650 BC, Egyptians were the first to use honey as a component in the topical treatment of wounds. 10 Honey is the most ancient wound dressing known, and it has continued to be used throughout the ages. 11 Dioscorides (c.50 AD) wrote of honey being 'good for sunburn' and 'for all rotten and hollow ulcers', and its usage has continued into present-day folk-medicine. It is used as a traditional therapy in Ghana for infected leg ulcers. 12 It is still being used as a dressing material for burn wounds, decubitus ulcers, gunshot wounds and wound dehiscence. It enhances auto debridement by absorbing oedematous fluid around the ulcer margins and promotes granulation tissue formation and epithelization. 13 This study was conducted over a period of one and half year and included 100 patients. The age group of patients was almost similar in the both the groups. These patients were divided into two groups, of which the first group (Group-A) dressing of chronic ulcer was done by honey and in the second group (Group-B) dressing of chronic ulcer was done by betadine (povidone iodine). In Group-A, 86% patient (43 cases) showed improvement in their wound, while 14% patient (7 cases) did not have any significant changes in their wound. In Group-B, 50% patients (25 cases) showed improvement of their wound, while 50% patients (25 cases) did not show any significant changes. Thus, honey had significantly better wound healing effect when compared with betadine. Several authors have reported that Honey enhances wound healing rate, compared to other conventional or topical application in a variety of clinical conditions, namely burns chronic wounds, infected surgical wounds and pressure ulcer.
In 1999 Krammer conducted a review of the clinical trials in which povidone iodine was used for cleaning, irrigating and dressing wounds. He concluded that povidone iodine did not effectively promote good wound healing and did not reduce bacteriological wound infection. 14 Our results with honey were similar to reports in literature showing rapid healing with the application of honey. 15 Moreover, Honey caused no adverse reaction. Hamdy et al.
reported that the number of microorganisms and bacterial species decreased by 50-100% by application of honey on chronic infected wounds. 16 Various authors reported mild or no pain at all during dressing change with honey as compared to other treatments. 17,18 In our study, percentage of the subjects in the honey dressing group achieved complete healing of chronic wound at the six weeks. Methi et al. reported similar finding by conducting a meta-analysis to evaluate the efficacy of topical application of honey in observational studies as well as in clinical trials in the treatment of wounds. Most of the subjects reported complete healing within 4-12 weeks in clinical trial and The median pain score (VAS) at the starting of study was 8.08 at honey dressing group and 8.02 at povidone-iodine dressing group. b The initial median surface area of the wounds 3.88 cm 2 in povidone-iodine dressing group and 4.07 cm 2 in honey dressing group. Table-1 19 The present randomised trial is unique in the sense that the salutary effects of honey have been combined with those of "moist occlusive" dressing. Occlusion of a wound cavity with semipermeable membrane retains the moisture of wound exudates and serves as a moisture retentive dressing. 20 Evaporation of exudates through an open dressing, namely gauze and cotton leads to cooling, desiccation, and dehydration of surface cells. The mitotic rate of healing cells is diminished at lower body temperature. 21 Moreover, the layer of dehydrated dead cells on the top of wounds cavity serves as a good food for microbes hampering healing. These adverse effects of open dressing are averted with the application of occlusive dressing. The occlusion can be achieved with the help of semipermeable polyurethane or hydrocolloid dressing. 21 A meta-analysis on trials comparing hydrocolloid occlusive dressing versus paraffin gauze and cotton dressing demonstrated significant better healing with occlusive dressing. The odd ratio of healing with hydrocolloid dressing was 1.72 compared to conventional paraffin gauze dressing (i.e. 72% more wounds healed completely with hydrocolloid dressing than with conventional paraffin gauze dressing. 21 This meta-analysis confirmed the beneficial effects of moisture retention occlusive dressing in terms of complete healing of chronic wounds. The good results observed in the honey treated group may be combined effect of honey and tegaderm occlusive dressing. Tegaderm offered an opportunity to inspect the wound cavity without the need for disturbing the dressing. It provides an occlusive environment in which honey could exert its full beneficial effect as entire honey poured into the cavity remained available to the healing cells. When honey application to a wound is covered with gauze or cotton, most of it gets absorbed in the gauze and is not available to healing cells. Moreover, polymorphs and macrophages immigrating into the wound cavity liberate growth factors essential for healing (i.e., epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor). These are retained in the wound cavity by an occlusive film like Tegaderm allowing them to exert their full biological activity. 22 In the conventional dressing with gauze and cotton, the wound cavity is deprived of large fractions of these essential molecules as they get absorbed in gauze and cotton, losing their biological activity.
In the western world, most wound clinics do not recommend the use of povidone iodine application on clean wounds. However, in India many physicians and nurses frequently use povidone even in clean wounds. The present study demonstrates that the rate of wound healing with povidone iodine and Tegaderm was much slower than achieved by the honey treated group.
CONCLUSION
Honey dressing is more effective as compared to povidone iodine dressing in achieving complete healing, reducing wound surface area and pain, and increasing comfort in subjects with chronic wounds. | 2019-06-13T13:22:43.153Z | 2018-12-01T00:00:00.000 | {
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18588992 | pes2o/s2orc | v3-fos-license | Requirement for vasoactive amines for production of delayed-type hypersensitvity skin reactions.
The skin sites of the mouse where delayed-type hypersensitivity (DTH) reactions are most easily elicited (foot pads and ears) are particularly rich in 5-hydroxytryptamine (5-HT)-containing mast cells. Since mice are deficient in circulating basophils, which play a role in at least some DTH reactions, we investigated the possibility that the mast cells were playing an important role in the evolution of the skin reactions of DTH in mice. We found that reserpine, a drug which depletes mast cells of 5-HT, abolished the ability of the mouse to make DTH reactions in the skin. The suppressive effect of reserpine could be partially blocked by monoamine oxidase inhibitors which prevent the degradation of 5-HT in the cytosol of the mast cell. Spleen cells of immune, reserpine-treated mice transferred DTH reactions to nonimmune mice normally, indicating that the reserpine treatment did not affect immune T cells. DTH reactions could not be transferred into reserpine-treated mice. We suggest that T cells are continually emigrating from the blood, through postcapillary venule endothelium, by a mechanism which does not depend on vasoactive amines. If they are appropriately immune and meet the homologous antigen in the tissue, they induce mast cells to release vasoactive amines which cause postcapillary venule endothelial cells to separate, allowing the egress from the blood of cells which ordinarily do not recirculate. The secondarily arriving vasoactive amine-dependent cells are responsible for the micro- and macroscopic lesions of DTH reactions. Chemotactic factors may also be involved in bringing cells to the DTH reaction sites but we propose that T-cell regulation of vasoactive amine-containing cells allows the effector cells to pass through the endothelial gates after they are called.
Injection of antigen into the dermis of the flank of an appropriately immunized rat, guinea pig, monkey, or man results, 24-48 h later, in the formation of an erythematous, indurated lesion . Similar skin testing of immunized mice generally fails to produce such lesions (1)(2)(3). The explanation for this particular difference between mice and men is unknown but there is reason to believe that it may not stem from differences in immunologically competent cells. Two observations support this view. (a) Appropriately immunized mice exhibit antigen-specific delayed-type hypersensitivity (DTH)' reactions when the site of elicitation is the foot pad (4) or the ear (5) . (b) Mice exhibit most other manifestations of cell-mediated immunity, in a normal fashion, despite their failure to produce DTH reactions in the flank skin. Thus, mice must have appropriately reactive T cells but there may be some difficulty in delivering the cells required for the production of DTH reactions to the flank skin . In support ofthis notion, it has been shown that ifperitoneal exudate cells are added to the eliciting dose of antigen placed in the flank skin the lesions that result are morphologically indistinguishable from those of classical DTH (2). Appropriate immunization and the inclusion of the specific antigen with the exudate cells are required for the production of these lesions. It thus appears that sensitized T cells may react with antigen in the flank skin of mice but that the subsequent migration of nonimmune cells, which is required for production of the lesions (6-11), does not normally ensue.
It has recently been shown that basophils comprise a significant portion of the inflammatory exudate in almost all types of DTH reactions of guinea pigs and man (12) and can play a functional role in these reactions (13) . Since basophils are important carriers of vasoactive amines, we considered it possible that the importation of these substances to the site of delayed reactions might play an important role in the development of the lesions. If this were true, it could explain why mice usually fail to exhibit delayed lesions in their flank skin, as this species is markedly deficient in circulating basophils (12,14). Production of * Supported in part by grants from U.S . Public Health Service no . AI-12211, AI-11707, AI-10947, CA-08597, NS-07436, and from the American Cancer Society, Inc. (IM-70A) .
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THE JOURNAL OF EXPERIMENTAL MEDICINE -VOLUME 142,1975 lesions in the foot pads and ears might result if these areas were particularly rich in mast cells which could substitute, at least in part, for recruited basophils. Therefore, we have examined the role of vasoactive amines in the production of DTH reactions in the foot pads of mice . Our results show that the skin of the mouse's foot pad contains many more serotonin (5-hydroxytryptamine)-containing mast cells, per unit weight, than does the flank skin and that pretreatment of mice, with the monoamine depleting drug reserpine, blocks the elicitation of DTH. This effect ofreserpine can itself be blocked by monoamine oxidase (MAO) inhibitors, confirming that the action of reserpine depends upon the intracellular release and catabolism of monoamines by MAO . It is known that the monoamine serotonin is the principal mediator of anaphylactic reactions in the mouse (15,16) . We suggest that this substance may also play an important role in the production of the lesions of DTH, perhaps by acting on the endothelial cells of the postcapillary venules (17) and permitting the egress of macrophages or their precursors from the blood into the sites of delayed inflammation . Macrophages which ordinarily do not pass through postcapillary endothelium, and thus do not recirculate, are the cells principally responsible for the swelling and induration of DTH reactions (6)(7)(8)(9)(10)(11) .
Materials and Methods
Mice . Mice were 6-to 8-wk old male BDF, (C57BL/6 x DBA/2) from the Jackson Laboratories, Bar Harbor, Maine. They were rested 1 wk in our animal facilities before use.
Immunizations
SHEEP RED BLOOD CELLS (SRBC) . Groups of five-six mice were immunized, under light ether anesthesia by intravenous injection of 0.2 ml of a freshly washed suspension of SRBC (0 .01% ; about 2 x 10 5 cells) in saline, or by separate subcutaneous 0.05-ml injections into the four proximal limbs of an emulsion containing (in equal parts) 10% SRBC and CFA (H37Ra, Difco Laboratories, Detroit, Mich .), fortified with 3 mg/ml ground mycobacterium tuberculosis .
Foot Pad and Ear Skin Testing
FOOTPAD. Mice immunized intravenously with 0.01% SRBC were tested 4 days later [this has been shown to be the time of optimal delayed foot pad responses produced by this mode of immunization (19)]. Separate groups of mice immunized with SRBC in CFA were tested at day 4 and day 10 . Freshly washed 1-wk old SRBC (20% in saline) were injected (0 .03 ml) into the ventral foot pad after foot pad thickness had been measured using a micrometer (Brown & Sharpe Mfg. Co ., No. Kingston, R. I.) . Subsequent 24-h foot pad thickness was compared to the original measurement and the percent change calculated .
EAR SKIN . Contact reactions of the ear skin were elicited by applying to each side of both ears a drop of freshly prepared 3% oxazolone in olive oil 7 days after immunization (5) or 0.2% DNFB in acetone-olive oil 5 days after initial immunization (18) . Micrometer-measured ear thickness at 24 hs was compared to ear thickness observed before contact testing, and percent change calculated . Student's t test was used to compute statistical significance (P < 0.05) .
Reactions of Passive Cutaneous Anaphylaxis (PCA) (20) . Mice were contact-sensitized with oxazolone and boosted with similar additional applications to the abdominal skin at days 7 and 14. Serum harvested at day 21 was diluted twofold in saline starting at 1 :20. 0.02 ml of four separate dilutions were injected intradermally into the shaved skin of groups of three to four Swiss mice anesthetized with ether. 2 h later 0.2 ml saline containing Evans Blue Dye (1/4%) and 0.5 mg oxazolone-human serum albumin (21) was injected intravenously. 30 min later mice were killed and the skin reflected to read the diameter and intensity of extravasated dye.
Drugs RESERPINE (5 MG/KG) . The amine-depleting drug reserpine (22,23) (Serpasil for parenteral injection, Ciba, Summit, N.J .) was injected as a single dose 6-12 h before testing of the skin or foot pad. Reserpine has a number of advantages as an experimental tool with which to manipulate monoamines . It has a long duration of action on amine storage, one which outlasts the presence of the drug in the animal . This permits possible direct effects of reserpine to be differentiated from the effects secondary to amine depletions. Reversal or antagonism of reserpine's action by MAO inhibitors permits a second check on drug specificity. Moreover, the well-characterized depletion of amines by reserpine is intrinsically a more specific approach than would be, for example, the use of antagonists which may have several actions, not all of which are known, and which may block some, but not all of the amine receptors; multiple 5-HT receptors have recently been recognized (24,25).
Determination of the Uptake of 5-HT SCINTILLATION COUNTING . (a) In the skin : Six mice were pretreated at 48 and 24 h with 6hydroxydopamine and with the MAO inhibitor pheniprazine . 30 min after the injection of pheniprazine the animals received 0.1 mCi of tritiated 5-HT ([ 311]5-HT in 0.1 ml saline ; serotonin binoxalate, New England Nuclear Corp . Boston, Mass ., 1.33 Ci/mmol) intravenously. 2 h later the mice were killed by cervical dislocation and skin was removed from the feet, flanks, and ears . Care was taken to avoid including cartilage with the samples of ear skin. Tissues were quickly weighed and homogenized in 5.0 ml of 70% ethanol. The resulting suspensions of tissue were allowed to stand overnight at 4°C. After centrifugation at 12,000 g for 20 min, aliquots were removed for chromatographic analysis and liquid scintillation counting . This procedure has been shown to extract better than 95% of the radioactive 5-HT from tissues (28) . Thin-layer chromatography done with butanol:acetic acid :water (12 :3 :5) or isopropanol:ammonia : water (10:1 :1) on cellulose (Eastman Chemicals) revealed that radioactivity measured in the ethanolic extracts was taken as an assay of the amount of [311]5-HT in the tissue and was expressed as dpm/g. Samples were dissolved in 15 ml of "Aquasol" (New England Nuclear Corp .) for liquid scintillation counting and corrected for quench by external standardization .
(b) In tumors: Mice carrying a mast cell tumor (P-815) were injected intraperitoneally with 100 mg/kg of pargyline to inhibit MAO. 1 h later each mouse was injected intraperitoneally with 0.1 mCi of [311]5-HT. Another two mice carrying the mast cell tumor were injected with 5 mg/kg of reserpine 4 h before injection of [ 311]5-HT. One of these animals was given pargyline as above and the other received no other drugs before injection of the isotope. 2 h after the injection of [311]5-HT all mice were killed, their abdomens opened and washed with 2 .0 ml of iced Krebs solution, and the tumor cells collected.
After collection in Krebs solution, the cells were pelleted by centrifugation at 1,200g for 10 min in a refrigerated centrifuge. The supernate was discarded, the cells were drained, and the pellets were weighed. 5 ml of 70% ethanol was then added to the cells. The cells were homogenized, allowed to stand overnight, centrifuged again as described above, and aliquots of the supernate were taken for liquid scintillation counting and chromatographic analysis .
RADIOAUTOGRAPHY . Experiments were designed to determine if the assumption that all of the tritiated 5-HT in the skin was localized to mast cells was valid. This was done by radioautography. Methods were the same as those described by Gershon and Ross (29,30).
Five mice were injected intravenously with 5.0 mCi of tritiated 5-HT creatinine sulfate (Amersham/Searle Corp ., Arlington Heights, Ill. ; spec act 5-18 Ci/mmol) . Injection volume was 0.1 ml . Pheniprazine was given to inhibit MAO 60 min before injection of the isotope. Segments of skin were removed after 2 h and fixed in iced 6.25% gluteraldehyde made hypertonic by the addition of 9% sucrose and buffered to pH 7.4 by 0.1 M phosphate buffer . Under these conditions of fixation, movement of [3 H]5-HT is abruptly halted (27,29). [ 3H15-HT is preserved in situ but its metabolites are not. After fixation for 2 h in gluteraldehyde, tissues were washed briefly in the sucrose-containing buffer solution and were postfixed for 1 h in 1% OSO4. The OSO. was buffered to pH 7 .4 with 0.1 M phosphate buffer containing 9% sucrose . The tissues were then rapidly dehydrated and embedded in epoxy resin (Epon 812) . Sections were cut at 0.5 Wm, placed on glass slides, and coated with Ilford L4 emulsion (diluted 1 :1) by dipping. The emulsion-coated slides were exposed for 1 wk in a dry, light-tight box under an atmosphere of 100% CO2. After photographic development and processing, slides were stained with toluidine blue for light microscopic examination. None of the processing of the fixed tissues extracts significant quantities of radioactive material (29)(30)(31).
Histochemical Demonstration of 5-HT . Tissue was examined histochemically for the presence of 5-HT by the formaldehyde-induced fluorescence technique of Falck and Hillarp (see 32 for refs .) . Treatment of the frozen-dried tissues with paraformaldehyde was modified according to the procedure of Fuxe and Jonsson (33) . Briefly, small pieces of tissues were quenched in melting Freon 22 that had previously been cooled with liquid nitrogen . Tissues were freeze-dried overnight in a Pearse-Edwards freeze drier, and then exposed at 80°C to formaldehyde gas generated first from paraformaldehyde equilibrated at 70% and then at 90% relative humidity . Tissues were embedded in hydroxybutyl methacrylate, sectioned at 3 Wm, and mounted on slides in nonfluorescent immersion oil under quartz cover slips for fluorescence microscopy . The microscope was fitted with a vertical illuminator (Leitz Ploem system, E . Leitz, Inc., Rockleigh, N.J .) . Identification of the fluorophore as that of 5-HT was done by microspectrofluorophotometry (Leitz-Schoeffel microspectrofluorophotometer). Activation maxima were at 385 and 420 nm and the emission maximum was at 520 nm . Controls included tissues heated as above at 80°C but not exposed to paraformaldehyde . Hairs fluoresced in the absence of formaldehyde treatment and was therefore due to autofluorescence and not to the presence of a monoamine.
Histochemical Demonstration ofHistamine. Tissue also was examined histochemically for the presence of histamine by the o-phalaldehyde-induced fluorescence technique of Ehinger and Thunberg (34) as modified by Cross et al . (35) . Freeze-dried tissue was embedded in paraffin, sectioned at 5 jLm, deparaffinized with xylene, exposed to o-phalaldehyde vapor at 100°C, and subsequently humidified . Sections were mounted in Entellan (Merck Chemical Div., Merck and Co ., Inc., Rahway, N.J .) and examined by fluorescent microscopy using a vertical illumination and a TK 400 dichroic beam splitting mirror and a K 430 suppression filter .
Results
Distribution of Mast Cells at Various Skin Sites in the Mouse . We studied mast cell distribution by several techniques : (a) After chemical sympathectomy for the prevention of [3H]5-HT uptake by adrenergic nerves, we measured the uptake of [3H]5-HT in various regions of the skin . The results (Table I) indicate that the concentration of mast cells, as determined by their ability to take up radioactive 5-HT, is in the order of foot greater than ear greater than flank.
The quantitative results obtained by scintillation counting were checked by radioautography to determine where in the skin the 5-HT had localized. More [3H]5-HT was given (50 times as much) to be certain that small amounts bound in unexpected locations would not be overlooked . The only labeled structures that we could find in any of the slides of skin were mast cells (Fig . 1) . These cells could be readily identified by their metachromasia in the toluidine blue-stained sections under the layer of photographic emulsion . The cells were very heavily labeled. No evidence of uptake of 5-HT by perivascular sympathetic nerves could be found . If animals were treated with reserpine (5 mg/kg) 1 day before injection of [3H15-HT, no labeled mast cells were found. These observations support the view that mast cells account for the uptake of labeled 5-HT by the skin. Therefore, the amount of tritiated 5-HT found in the skin probably is a function of the number of activity of the mast cells in the area .
(b) Independent confirmation ofthe quantitative data derived from studies of the uptake of [3H]5-HT, suggesting that mast cells were most numerous in the skin of the foot, was sought . Mast cells of mice and rats are known to contain 5-HT and histamine (see 14) . Therefore, 5-HT and histamine was demonstrated in sections of the skin histochemically in order to visualize mast cells and to estimate their numbers. By means of formaldehyde (5-HT) and o-phalaldehyde (histamine)-induced histofluorescence, many brightly fluorescent mast cells could be seen in sections of the skin of the foot, especially on the dorsal surface clustered around autofluorescent hair follicles (Fig . 2). Far fewer fluorescent mast, cells were found in sections of flank skin (Fig . 3), while ear skin had intermediate numbers. These results confirm those above; that mast cells in the skin which contain 5-HT are far more numerous in the skin of the feet and somewhat more numerous in the skin of the ears than of the flank, and that histamine-containing mast cells have the same distribution . Mast cells identified by numerous cytoplasmic metachromatic granules in paraffin sections viewed with an ordinary light microscope after Helly's fixation and Giemsa staining (36) were similarly distributed.
The Effect of Reserpine on the Uptake of 5-HT . Radioautographic observations indicated that pretreatment of mice with reserpine totally ablated the capacity of the mast cells to take up and/or store 5-HT . We also studied the ability of reserpine to block the uptake of [3H]5-HT by P815 mastocytoma cells. As a specificity control for the uptake of the 5-HT by the mastocytoma cells, we used a different mouse tumor, sarcoma I. The results of this experiment (Table II) show that the mast cell tumor took up more 5-HT than did sarcoma I cells.
Reserpine antagonized the ability of the tumor cells to accumulate 5-HT and this antagonism was partially overcome by the administration ofthe MAO inhibitor pargyline.
Effect of Reserpine Pretreatment on the Ability of Mice to Make a DTH Response to SRBC . Mice were immunized intravenously with 0.2 ml of a 0 .01% suspension of SRBC . Their ability to mount a DTH response was tested 4 days later by injecting 0.03 cc of SRBC into the foot and measuring the increase in foot pad thickness 24 li later. Some of the mice were treated with reserpine alone, some with reserpine plus the MAO inhibitor nialamide, some with 5hydroxydopamine as a control for the effects ofreserpine on norepinephrine (27), and some with burimamide, a blocker of histamine-2 (H2) receptors (26) [antihistamines which work on H, receptors are known not to affect reactions of delayed hypersensitivity (37) and in any case the mouse is extraordinarily resistant to histamine (see 16)]. The results of this experiment (Table III) show that reserpine treatment abrogated the DTH response occurring 24 h after challenge with antigen, that the MAO inhibitor nialamide partially reversed this suppression, and that the depletion of norepinephrine alone and H2 blocking were without effect . We subsequently found that delaying the injection of an MAO inhibitor until 6 h after administration of reserpine fails to reverse the effect of reserpine on DTH reactions but does reverse the central nervous system effects.
FIGS . 2 and 3. Formaldehyde induced fluorescence of serotonin (5-HT) in the skin of mice . 5-HT-containing cells were quite numerous in the dorsal foot skin (Fig . 2), and were localized to the upper dermis where mast cells were most numerous as judged by light microscopy . The cells at the lower right demonstrate that 5-HT was localized to the cytoplasm (x 100) . Fluorescent cells were noted only occasionally in the flank skin (Fig. 3) .
[(o-phalaldehyde-induced fluorescent cells (histamine-containing) were similarly distributed).] 738 We tested the possibility that the low dose SRBC immunization schedule we used was not truly representative of a DTH response . To do this, we immunized mice with SRBC in CFA and tested the ability of reserpine to block the delayedhypersensitivity response in the footpad 4 and 10 days after immunization. The results (Table IV) show that reserpine could block the delayed response to this form of immunization as well as it could to the low dose immunization .
Effect of Reserpine on Contact Reactions . We also tested the ability of reserpine to inhibit reactions of contact hypersensitivity occurring on the mouse ear. The results (Table V) show that reserpine was also able to inhibit these forms of DTH, although the inhibition was not as complete as in the SRBC system .
Effect 3 -t 2 (1+) 0 0 Reserpine + 0 0 0 nialamide of DTH. This latter point was of particular interest since reserpine is known to act by causing stored amines to leak from the granules of mast cells into the cytosol, where they are inactivated by MAO (38) . Since inhibition of MAO after reserpine would lead to the accumulation of free amine in the cytosol of mast cells, this suggested that some form of nongranule release of amines might be occurring. We therefore tested whether reserpine could inhibit PCA reactions which are known to be dependent upon exocytosis of mast cells granules (39) and to see whether nialamide could reverse that form of inhibition . The results of three pooled experiments (Table VI) show that reserpine significantly inhibited PCA reactions, but in contrast to the DTH reactions, nialamide was now unable to reverse this inhibition .
Effect of Adrenalectomy on the Ability of Reserpine to Block Reactions of Delayed Hypersensitivity . Although our results show that reserpine treatment affects the ability of mast cell granules to bind and store 5-HT, we considered the possibility that this was not causatively related to its ability to block reactions of DTH but that there might be an indirect action . We had ruled out effects secondary to reserpines tranquilizing action by the differential protection afforded on behavioral effects and DTH by delayed treatment with MAO inhibitors . Another indirect action could come from stress and the subsequent release of corticosterone from the adrenals . We therefore tested the ability of reserpine to inhibit DTH reactions in adrenalectomized mice . The results of this experiment (Table VII) show that adrenalectomy was without significant effect on the DTH in either untreated or reserpine-treated mice . Therefore, nonspecific stress was not a significant factor in our results.
Effect of Adoptive Transfer of Immune Spleen Cells from Reserpine-Treated Mice . We performed a more direct test for secondary effects of reserpine treatment by doing adoptive transfer experiments. We immunized mice with SRBC and treated some of them on day 4 with reserpine. 8 h after injection of reserpine, we harvested the spleens and transferred the resultant cell suspension (one spleen : two recipients) to normal nonimmune syngeneic mice, some of which had been pretreated with reserpine at the same time as the donor mice . The results (Table VIII) show that treatment of donors with reserpine abrogated their ability to mount a DTH response (line 4). However, spleen cells of the reserpine-treated donors, who themselves could not make a DTH response, were able to transfer DTH to normal recipients (line 3), as well as spleen cells from donors who had not been treated with reserpine (line 1) . If however, the recipients had been treated with reserpine, the transfer of immune spleen cells failed to convey the ability to mount a DTH response to the recipient (line 2). Therefore, reserpine acts on cells of the host, not on the immunocompetent cells of the donor.
Discussion
We would like to set forth some previously made observations and those elucidated in the present study, and from them put forth a hypothesis to explain certain elements of delayed hypersensitivity reactions and their relationship to other immunological phenomena. Pertinent observations made in previous studies include the following: (a) mice are relatively deficient, compared to other animals, in their ability to make DTH reactions in the flank skin (1-3); (b) they nevertheless make excellent DTH reactions when the eliciting antigen is placed in the foot pad (3,4) or on the ear (3, 5); (c) mice are relatively deficient in basophils, circulating cells which carry vasoactive amines (12,14), and these cells are often found in the cellular exudate in DTH reactions of other mammals (12) ; (d) vasoactive amines allow intravascular contents to pass into the tissue by causing the endothelial cells of postcapillary venules to contract and separate (17); (e) the predominant recirculating lymphocyte found in the mouse thoracic duct is a T cell (40), indicating that these cells are especially equipped to leave the blood and enter the lymph; (f) T cells play an important role in the recruitment and/or entrapment of cells from the circulation to lymphoid tissues draining sites of exogenously administered antigen (41).
Observations made in the present study include the following: (a) mast cells which contain vasoactive amines are particularly numerous at skin sites in the mouse where DTH reactions are most easily elicited ; (b) vasoactive amine depletion by reserpine severely inhibits various DTH reactions; (c) the techniques used for depletion of vasoactive amines do not alter the ability of T cells of treated mice to adoptively transfer DTH reactions to normal recipients. Thus, the site of action of vasoactive amine depletion is not at the level ofthe effector T cell; (d) pharmacological treatments which cause depletion of vasoactive amines from mast cells granules, but which favor accumulation of free amine in the cystosol, (reserpine plus MAO inhibitors) eliminate reactions of passive cutaneous anaphylaxis but not those of delayed hypersensitivity.
To account for these observations, we propose that T cells are especially equipped to traverse the endothelium of postcapillary venules, which they continually do . If they are appropriately immune and meet the specific antigen outside of the circulation, they release a factor that causes nearby mast cells and/or basophils to release their vasoactive amines . If basophils are absent, mast cells acquire added importance . The released amines cause the endothelial cells of the postcapillary venule to separate, allowing the rapid egress from the blood of cells which do not normally recirculate. Nonrecirculating cells coming from the bone marrow are the predominant cell type that causes the swelling and induration of a delayed hypersensitivity reaction (6)(7)(8)(9)(10)(11) . There probably is an additional element of a T-cell-released chemotactic substance or substances which attract the appropriate cells to the site (42) . However, the release ofthese chemotactic substances is insufficient in itself to permit the secondarily arriving cells to enter the reaction site, unless the walls of the postcapillary venules have been acted upon by vasoactive amines .
This T cell-to mast cell-to postcapillary venule reaction is also probably very important in the general regulation of lymphocyte traffic. [We have evidence that depletion of 5-HT from the mouse abrogates the antigen-driven trapping of lymphocytes in the lymph nodes but not in the spleen (Gershon and Kondo, unpublished observations) .] This may be why workers studying the mode of entrance of lymphocytes into nonantigen-stimulated lymph nodes find that cells go through, rather than between, endothelial cells (43) . Those who study the mode of entrance in lymphoid tissue of the gut where there is constant antigenic stimulation have found that the cells go between the endothelial cells of the postcapillary venule (44) . There may be a continuing antigen-to T cell-to mast cell reaction going on in lymphoid tissue draining the gut, which accounts for the different mode of circulation.
Although our results do not bear directly on the mechanism by which T cells cause mast cells to release their vasoactive amines, they do suggest that the mechanism is different than that of PCA reactions, which involve exocytosis of amine storage granules (39) . MAO inhibition should not affect any response which is mediated solely by granule release because oxidative deamination takes place in the cell cytosol rather than in the granule (22,23). Reserpine causes 5-HT to leak out of granules into the cytosol where inactivation by MAO takes place (22,23,38,45). Thus, MAO inhibition in theory should only protect reserpine-inhibited reactions in which the cytosol as well as the granules is released. Since MAO inhibition partly protected the delayed response of mice treated with reserpine and failed to protect the PCA response, it seems probable that mast cell cytosol is released in delayed reactions. Cytosol release would occur if the mast cell was killed or damaged by the T cell . T cells of appropriately immunized mice are known to migrate to the skin and to release cytotoxic factors in the presence of specific antigen (2,46). These released factors are toxic for macrophages and may be related to the lymphokine called lymphotoxin (47) . If lymphotoxin was toxic for mast cells it could be the important link between the T cell and the firing of the mast cell . A number of points raised in the above hypothesis are easily testable . Some of them have already been confirmed and others we are in the process of testing.
Several remarks of clarification are in order. Although all the evidence we have presented suggests that 5-HT is the main vasoactive amine involved in the mediation of the reactions of DTH in the mouse [as it is in anaphylactic reactions (16)], we wish to stress that participation by other amines or factors has not been ruled out. There is ample evidence to indicate that different vasoactive amines can interact with one another in the production of vascular alterations (48) . In addition, the role of 5-HT carrying platelets (49) in these reactions must be considered . Clearly, a great deal more work is required before the chemical basis for our observations can be soundly and thoroughly elucidated. The present report serves simply to implicate the importance of vasoactive amine release in DTH. This fact has heretofore not been fully appreciated, although the early work of Voisin was highly suggestive in that it showed that vascular permeability changes occurred during the evolution of DTH reactions (50) . Perhaps one reason the establishment of the role of vasoactive amines in DTH has been delayed is that the amines and the interactions between them and their various cell receptors are so complex. For example, the nature of histamine (26) and serotonin (24,25) receptor subtypes has only been recently recognized . The mouse is probably an exceptional model for these studies since histamine mediation of vascular reactions in this species is so much less important than in others (16), allowing us to manipulate the response by affecting the storage of 5-HT . Obviously, we must try to duplicate our results in other species but, from our preliminary attempts to do so in the guinea pig, it seems that this will be a more formidable task than it was in the mouse. We should also point out that there have been a number of reports on the effect of reserpine on the immune response (see 51). These previous studies, however, all used multiple injections of reserpine, which led to cachexia and stress ; therefore it is difficult to relate them to the findings we have made .
Finally, we would like to comment on what relationship our findings may have to other areas of cellular immunology such as tumor rejection . It has been shown that some tumors can elicit a very strong cell-mediated immune response against themselves and yet continue to grow progressively in the face of this immune response until death of the host; a phenomenon referred to as concomitant immunity (52) . Although the primary tumor is not rejected, subcutaneous or intravenous grafts of isolated tumor cells fail to take in the animal with the progressively growing tumor. In addition, the concomitant immunity is able to prevent viable circulating tumor cells in the tumor-bearing host from forming metastatic deposits. Although quantitative factors are certainly involved, it has been shown that, over and above these, the tumor acquires resistance to the hosts immune response when it becomes stromatized (53). We suggest that a possible mechanism accounting for the phenomenon of concomitant immunity is that tumors may lack (a) mast cells, or (b) postcapillary venules and thus may be isolated to some degree from the immune killer cells they have engendered in the host in which they are growing . A parallel situation has been reported in experiments on allergic encephalomyelitis (54,55) . The work we have presented highlights an important, under-investigated area ofimmunology : delivery of cells to sites of reaction . The finding that tumorbearing hosts contain lymphocytes which are able to kill tumor cells in vitro but which have not been demonstrated to have in vivo effects, indicates that the problem of delivery may be of broader importance in health care then has heretofore been generally realized .
Summary
The skin sites of the mouse where delayed-type hypersensitivity (DTH) reactions are most easily elicited (foot pads and ears) are particularly rich in 5hydroxytryptamine (5-HT)-containing mast cells. Since mice are deficient in circulating basophils, which play a role in at least some DTH reactions, we investigated the possibility that the mast cells were playing an important role in the evolution of the skin reactions of DTH in mice . We found that reserpine, a drug which depletes mast cells of 5-HT, abolished the ability of the mouse to make DTH reactions in the skin. The suppressive effect of reserpine could be partially blocked by monoamine oxidase inhibitors which prevent the degradation of 5-HT in the cytosol of the mast cell . Spleen cells of immune, reserpinetreated mice transferred DTH reactions to nonimmune mice normally, indicating that the reserpine treatment did not affect immune T cells. DTH reactions could not be transferred into reserpine-treated mice.
We suggest that T cells are continually emigrating from the blood, through postcapillary venule endothelium, by a mechanism which does not depend on vasoactive amines . If they are appropriately immune and meet the homologous antigen in the tissue, they induce mast cells to release vasoactive amines which cause postcapillary venule endothelial cells to separate, allowing the egress from the blood of cells which ordinarily do not recirculate . The secondarily arriving vasoactive amine-dependent cells are responsible for the micro-and macroscopic lesions of DTH reactions . Chemotactic factors may also be involved in bringing cells to the DTH reaction sites but we propose that T-cell regulation of vasoactive amine-containing cells allows the effector cells to pass through the endothelial gates after they are called . | 2014-10-01T00:00:00.000Z | 1975-09-01T00:00:00.000 | {
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253229102 | pes2o/s2orc | v3-fos-license | Characterization of novel bacteriocin PB2 and comprehensive detection of the pediocin gene ped-A1 from Pediococcus pentosaceus PB2 strain isolated from a sorghum-based fermented beverage in Nigeria
Highlights • Novel bacteriocin PB2 was obtained from Pediococcus pentosaceus PB2 strain isolated from fermented sorghum beverage in Nigeria, Pito.• The PB2 isolate exhibited antagonistic activity against E. coli ATCC 25922 and L. monocytogenes ATCC 15313.• The specific activity, purification fold, and recovery yield of bacteriocin PB2 from Carboxymethyl-Sephadex C-50 are 2.65 U/mg, 12.62 and 13.3% respectively.• The bacteriocin PB2 is a proteinous bacteriocidal agent with a molecular weight of 4.87 kDa, with optimum activity at 40 °C and pH 5.0. Bacteriocin PB2 lost its activity on treatment with proteinase K and exposure to UV radiation (after 6 h) but was observed to have stable activity in the presence of organic solvents.• The amplicon size of Pediococcus pentosaceus PB2 was 727 bp. P. pentosaceus was found to harbor two plasmids targeted from primers designed from operon encoding pediocin PA-1 gene. The molecular sizes of the plasmids are 0.9 and 1.2 kb. The curing analysis showed that the bacteriocidal activity of P. pentosaceus is derived from the presence of the plasmid present.
Introduction
Sorghum (Sorghum bicolor) is a cereal for the production of a good number of common staple foods in West Africa. In the international market, especially in the United States and Japan, sorghum has gained headway as it is gluten-free and possesses resistant starch, high fiber content, and bioactive components with antioxidant properties [1]. According to the Systematic Survey of Agricultural Production, in Brazil, the number of tons of cereal produced increased by more than 15% between 2018 and 2019 [2]; in the same country, it is being used as a nutrition source for animals [1]. Chadalavada et al. [3] reported sorghum as one of the staple foods to be drought tolerant, one that can be grown in arid and semiarid regions of the world. In Nigeria, sorghum is grown in the far Northern states under semi-arid conditions-Adamawa, Bauchi, Benue, Borno, Gombe, Jigawa, Kaduna, Kano, Katsina, Kebbi, Kogi, Kwara, Nasarawa, Niger, Plateau, Sokoto, Taraba, and Zamfara States; it is used for the production of fermented foods and beverages such as Koko, Furo-furo, Maasa, Fura, Ogi-baba, and Pito.
In our previous study, we determined microorganisms associated with Pito processing [4]. They were isolated and identified utilizing microbial, biochemical, and genomic techniques to be Lactobacillus plantarum and Pediococcus pentosaceus [4]. The aforementioned organisms are examples of lactic acid bacteria (LAB) [4]. LAB has a long history of application in fermented foods because of its beneficial influence based on its nutritional, organoleptic, safety, and preservative characteristics [4][5][6][7][8]. The involvements of LAB in Pito and similar traditional sorghum-fermented foods in Nigeria like Ogi-baba is based on the spontaneous fermentation process; a deliberate addition of the isolated microorganisms as starters to the food matrix would result in a high degree of control over the fermentation process to yield a more standardized end product. During fermentation, LAB displays a range of biochemical and metabolic activities such as the synthesis of organic acids and bioactive molecules such as ethanol, formic acid, fatty acids, hydrogen peroxide (H 2 O 2 ), and diacetyl [4]. The screening of LAB has revealed that besides metabolic activities, they may also exhibit probiotic potentials [4,9,10]. This class of bacteria produces inhibitory substances that impede the growth and survival of other bacteria. Strains of LAB that display antimicrobial activities usually produce bacteriocin that inhibits the growth of pathogenic and food spoilage microorganisms, thereby ensuring the safety of the product [11].
These bacteriocins are known to be ribosomal-synthesized, small, heat-stable antibacterial peptides with diverse modes of bactericidal activities against genetically related strains and species [12,13]. As established, most LAB are bacteriocinogenic, and may be isolated from milk, other dairy products, sorghum, and its products [4,14]. The nomenclature of bacteriocin is sometimes derived from its source and mechanism. Colicin is the name given to bacteriocins from Escherichia coli, wamericin is derived from Staphylococcus wameri, and bacteriocins from LAB are designated as lantibiotics [9]. Also, bacteriocin isolated from Pediococcus spp. is called pediocin. Pediocin AcH/PA-1 has been reported to be the most studied bacteriocin (non-modified, non-lantibiotic peptides) [9]. This further gives an insight that the bacteriocidal expression of P. pentosaceus strains would make them suitable as preservatives for foods, crops, and livestock [15]. Biological treatment towards preservation may sustain the quality and general safety of these products instead of chemical treatment. P. pentosaceus also inhibits enteric pathogenic bacteria, improves gastrointestinal tract microflora balance, and adheres to the mucosal surfaces of hosts [4].
In addition, when our research group previously isolated and identified P. pentosaceus PB2 from the well-known Nigerian Sorghum fermented products, Pito and Ogi-baba [4], the same strain was deposited in GenBank (NCBI KP883297) and submitted to the Laragen Incorporation, 10601 Virginia Ave, Culver City, CA90232, California, USA. We found P. pentosaceus PB2 to be acid tolerant, bile salt tolerant, lactic acid homofermenter, β-galactosidase producer, resistant to Oxacillin 5 μg, Streptomycin 10 μg and Tetracycline 25 μg and Chloramphenicol 25 μg [4]. Also, P. pentasoceus PB2 produced antibacterial chemical agents like lactic acid, diacetyl, and hydrogen peroxide. It also exerted its bactericidal effect on other bacteria, namely, Listeria sp., Bacillus sp., Staphylococcus sp., E. coli, H. pylori, Pseudomonas sp., Klebsiella sp., and Salmonella sp [4]. As a result of the bacteriocidal activity of some bacteria, bacteriocin production is now becoming a viable therapy option for both multidrug-resistant and chronic illnesses [16].
Previous research works have primarily shown the bacteriocidal effect of other strains of Pediococcus isolated from other sources but not from Pito and the PB2 strain. Mathys et al. [9] first reported bacteriocin produced by a human intestine Pediococcus acidilactici isolate, then a new real-time Polymerase Chain Reaction (PCR) assay was successfully developed to indicate the large distribution of pedA-containing strains in newborn faeces samples. Halami et al. [17] purified and characterized bacteriocin from Pediococcus pentosaceus ACCEL from vegetable sources. Recently from a characterization study, it was highlighted that the antimicrobial peptide from Pediococcus pentosaceus N33 strain isolated from pickles contained carotenoid pigments displaying good inhibitory effects against Brevibacillus brevis and Lysinibacillus fusiformis strains [16]. Also, Pediococcus pentosaceus NCDO 990 was found to exert similar effects in reducing the growth of Aeromonas hydrophila CAIM 347 at 12 h of interaction [18]. This manuscript presents the antimicrobial activity of P. pentasoceus PB2, isolated from the sorghum-fermented food, Pito, while detecting the pediocin gene ped-A1. There is currently no report in the open literature on the characterization of bacteriocin PB2 and the detection of the pediocin gene ped-A1 from Pediococcus pentosaceus PB2 strain obtained from Pito.
This work intends to provide a foundational basis for further investigation to improve the quality and medical applicability of the sorghum-fermented product. Results from this study can be instrumental in addressing the increasing rate of antimicrobial resistance (AMR) in Sub-Saharan Africa and globally. For a long time now, practitioners of traditional medicine administer Pito in the treatment of diseases (like digestive tract diseases including infectious diarrhea and inflammatory digestive tract diseases) which are now difficult to treat as a result of AMR. Also, computational studies elucidating the inhibitory potentials of these peptides against protein targets of disease-causing microbes could be done to further gain insights into the mechanism of inhibition from a molecular scale [19,20]. The findings from this paper would be of great relevance to indigenous and international researchers, pharmaceutical, food and nutraceutical industries, medicinal chemists, pharmacologists, pharmacists, medical practitioners, and policymakers in the area of food, protein, and probiotics.
Bacterial strains
The laboratory probiotic strain Pediococcus pentosaceus PB2 used in this study was isolated from Pito as described in our recent finding [4]. The PB2 strain was identified using genomic, biochemical, and microbial tools. It was then deposited in GenBank (NCBI KP883297) and submitted to the Laragen Incorporation, 10601 Virginia Ave, Culver City, CA90232, California, USA [4]. Two reference strains, Escherichia coli ATCC 25922 and Listeria monocytogenes ATCC 15313, obtained from Microbiologics, Medimark Europe, France, were also used.
Screening for bacteriocin production
Selected Pediococcus pentosaceus PB2 isolates were cultured in MRS broths and screened for bacteriocin production according to the method described by Van Reenen et al. [21]. The isolates were grown in MRS broths with lactose as carbon source at 37 • C for 18 h. The cell-free supernatant (CFS) was obtained by centrifuging at 10,000rpm for 5 min at 4 • C, and was adjusted to pH 7.0 with 1 M NaOH. Antimicrobial activity was monitored by using the agar-well diffusion test method [22]. All strains were then stored at -4 • C.
Ammonium sulphate precipitation of bacteriocin
Ammonium sulphate NH 4 (SO 4 ) 2 was added gently to the CFS to obtain 80% saturation with continuous stirring using a high-speed magnetic stirrer at 4 • C. The ammonium sulphate precipitated protein which was obtained by centrifuging at 14,000 rpm for 30 min. The obtained precipitate was collected and dissolved in sterile MiliQ water. Dialysis tubing (Spectrum Labs, USA) was utilized to dialyze samples for 8 h at 4 • C against phosphate buffer. The dialysate was then stored at − 20 • C overnight.
Carboxymethyl-sephadex G-50 column chromatography purification
The dialyzed sample was passed through Carboxymethyl-Sephadex G-50 Column Chromatography. Elution was done by the gradient of 10 mM phosphate buffer and 0.1 -1 M NaCl buffer (pH 7.0 ± 0.2). Fractions were collected at a flow rate of 0.4 ml/min and were monitored at 280 nm using the UV spectrophotometer for the activity [23].
Protein estimation and peptide assay
The biuret method was used to determine the protein level of the bacteriocin samples obtained after the purification process (HiPer R Protein Estimation Teaching kit). Agar well diffusion method was used to determine the antibacterial activity of the bacteriocin samples against E. coli ATCC25922 [24]. Antibacterial activity is measured in arbitrary units per milliliter (AU/ml). One arbitrary unit is defined as the reciprocal of the highest dilution that exhibits a minimum detectable zone of inhibition [25]. The activity, yield, and fold purification of bacteriocin obtained after various purification steps were also calculated.
Determination of molecular weight of bacteriocin PB2 by tris-tricine SDS-PAGE
The sample depicting a single peak protein from the CM-ion chromatography was subjected to Tris-Tricine SDS-PAGE analysis to determine its molecular weight [26,27]. The purified protein along with molecular weight markers (Dual Xtra standards, BIORAD, USA) was initially electrophoresed at 40 V for 30 min, thereafter at 100 V for 120 min. After electrophoresis, the gel was stained with Coomassie® Brilliant Blue and destained by washing overnight with a mixture of acetic acid-methyl alcohol-water (5:5:1 v/v).
Determination of the effect of different temperatures on bacteriocin PB2
The effect of temperature on the bacteriocin activity was determined by incubating the purified bacteriocin at pH 4.5 at 20, 30, 37, 40, 60, 80, and 100 • C, respectively for 2 h [28]. After treatment, the bacteriocin PB2 samples were kept at room temperature for 30 min and were then tested for antimicrobial activity against reference strains by using the agar diffusion test [22].
Determination of the effect of different pH on bacteriocin PB2
In determining the effect of pH on the activity of bacteriocin PB2 the pH of the purified bacteriocin PB2 was adjusted from 2.0 to 10.0. After 2 h of incubation at room temperature, the samples were tested for antimicrobial activity against reference strains by using the agar diffusion test [22].
Determination of the effect of enzymes and detergents on bacteriocin PB2
To the bacteriocin PB2, 1% (w/v) detergent including sodium dodecyl sulphate (SDS), urea, ox-gall, and hydrolytic enzymes including proteinase K and RNase A was separately added. The untreated bacteriocin was considered as a control, and its activity was taken as 100%. All reactions containing detergents were incubated at 37 • C for 6 h, then tested for antimicrobial activity against reference strains by using the agar diffusion test [22]. Also, the bacteriocin PB2 was adjusted to pH 5.0 with 1 M NaOH. A 1 ml aliquot of the CFS was incubated for 2 h on adding 1 mg/ml and 0.1 mg/ml of each of proteinase K and RNase (Sigma). The bacteriocin activity was assayed against reference strains by the agar-well diffusion method [22].
Determination of the effect of various organic solvents on bacteriocin PB2
The bacteriocin PB2 was treated with 50% v/v organic solvents including ethanol, phenol, acetone, chloroform, and isoamyl alcohol. The mixture was incubated at 37 • C for 2 h. After the incubation, the solvent was evaporated by aeration for 30min, then the antimicrobial activity against the reference strains was determined using the agar-well diffusion assay [22].
Determination of the effect of ultraviolet (UV) radiation on bacteriocin PB2
The bacteriocin PB2 from the isolate was placed in a sterile glass and exposed to short-waive UV light at a distance of 30 cm [22]. The duration of exposure to UV radiation ranged from 2 to 12 h. At 2 hr intervals, bacteriocin activities were analyzed by the agar-well-diffusion method against the different reference strains.
Determination of genes encoding bacteriocin production
2.4.1. Screening for the presence of bacteriocin gene P. pentosaceus PB2 extrachromosomal DNA elements were isolated using a modified approach based on Anderson and McKay [29] for small-scale plasmid separation. A 9.5 μl aliquot of mutanolysin (1500 U ml-1 Sigma-Aldrich Chemie GmbH) was added to the lysis solution (solution B) for cell lysis, and the plasmid DNA was resuspended in 1 x TE buffer. Finally, 10 g RNase A was used to degrade the RNA (Sigma-Aldrich Chemie GmbH). Bacteriocin gene-specific primers, Pedpro (5 ′ -CAA GAT CGT TAA CCA GTT T-3 ′ ) and Ped 1041 (5 ′ -CCG TTG TTC CCA TAG TCT AA-3 ′ ) were designed from the operon encoding pediocin PA-1 (accession number M83924) and synthesized by Genosys Biotechnologies (Europe) Ltd (Cambridgeshire, United Kingdom) [30]. Bacteriocin PB2 genes were amplified from the extracted genomic DNA of P. pentosaceus PB2 with the identified gene-specific primers. PCR reactions were performed using a BioRad® PCR (BioRad Thermal Cycler, USA). Amplification conditions were as follows: initial denaturation at 94 • C for 5 min, 35 cycles of 5 min at 94 • C, and 10 s at 61 • C, followed by an increase to 72 • C for 2 min. The final extension of the amplified product was at 72 • C for 75 min [43]. The obtained amplicons were purified with a QIAquick PCR Purification Kit (Qiagen), following the manufacturer's instructions, and submitted to sequencing at the Laragen Inc. Culver City California. The amplified product was visualized in a 1.2% (w/v) agarose gel stained with ethidium bromide.
Curing conditions
Ethidium bromide at a concentration of 125 µg/ml was added to bacteriocin-producing pediococcal cells to exert environmental pressures on cells to favor plasmid loss at a growth temperature of 37 • C for 48 h. After incubation time, the samples were tested for loss of bacteriocin phenotype (PB2-). Strains found to be PB2-were subjected to plasmid analysis along with PB2+ strains as controls. PB2-variants of P. pentosaceus termed plasmid-free cells were inoculated into the overnight culture of reference isolates.
Statistical analyses
Results from various sets of experiments were statistically examined by determining the mean, standard deviation (SD), and standard error (SE) from a minimum of three observations whenever necessary. All the statistical analysis was carried out using GraphPad Prism version 5.0 at a significance level of p ≤ 0.05.
Screening of bacteriocin production
Treatment with the CFS of P. pentasoceus PB2 showed inhibitory activity against the reference strains. Fig. 1 illustrates that the CFS of the isolate PB2 isolated from Pito exhibited antagonistic activity against the E. coli ATCC 25922 and L. monocytogenes ATCC 15313 compared to the LAB, L. plantarum which did not inhibit the activity of E. coli ATCC 25922.
Carboxymethyl-Sephadex G-50 column chromatography purification
For purification of bacteriocin, the precipitated protein was subjected to ion-exchange/gel-filtration chromatography using Carboxymethyl-Sephadex G-50 and eluted with 10 mM phosphate buffer, then with gradient salt (0.1 -1 M NaCl; pH 7.0 ± 0.2). The elution profile depicts four distinct peaks at 280 nm; of the four peaks which are proteins, the second peak (fractions showed maximum activity at 600 nm (Fig 2). Subsequently, the pooled fraction from the second peak was used in further characterization. The activity of bacteriocin PB2, yield and fold purification obtained after various purification steps are provided in Table 1. MIC = minimum inhibitory concentration
Determination of molecular weight of bacteriocin PB2 by tris-tricine SDS-PAGE
For determining the molecular weight of bacteriocin, the sample collected from Carboxymethyl-Sephadex G-50 was run on Tris-Tricine SDS PAGE. A distinct band of 4.87 kDa was seen after Coomassie brilliant blue staining. (Fig. 3) Further, to confirm the purity of the protein band corresponding to 4.87 kDa, the crude extract was overlaid on a different lane 3 of the gel in situ and the results are shown in Fig. 3.
Effect of different temperatures on bacteriocin PB2
The effect of temperature ranging from 20 • C to 100 • C on the antibacterial activity of the bacteriocin against reference strains, E. coli and L. monocytogenes, was studied and the results are given in Fig. 4. The effect of temperature was observed to have similar patterns in the reference strains; however the bacteriocin PB2 had higher activity against L. monocytogenes. It was observed that the bacteriocin PB2 was found to be heat sensitive as it could retain the antibacterial potential being treated between 30 and 80 • C (Fig. 4). However, at temperature regimes of 20 • C and 100 • C, the bacteriocin PB2 was observed to be of least activity. At 40 • C, the optimum activity of the bacteriocin was observed.
Effect of different pH on bacteriocin PB2
The influence of pH ranging from 2 to 10 on the activity of bacteriocin PB2 against reference strains was studied and the results are given in Fig. 5. The effect of pH was observed to have similar patterns in the reference strains. The bacteriocin PB2 had higher activity against E. coli through varying pH. Bacteriocin PB2 retains its antibacterial property at pH 3.0 through pH 9.0. Bacteriocin PB2 demonstrated the maximum bacterial activity at pH 5.0 against E. coli a and L. monocytogenes. However, at extreme pH 10 bacteriocin PB2 lost its activity.
Effect of detergents and enzymes on the bacteriocin PB2
The effect of detergents and enzymes on the activity of the bacteriocin PB2 against the reference strains was determined and the results are shown in Fig. 6. It was observed that the bacteriocin lost its antibacterial activity on treatment with Proteinase K. Bacteriocin PB2 demonstrated stability on treatment with SDS, urea, Ox-gall, and Rnase (Fig. 6).
Effect of various organic solvents on the bacteriocin PB2
The effect of various organic solvents on the activity of the bacteriocin against reference strains was determined and the results are shown in Fig. 7. It was observed that the activity of bacteriocin was stable on treatment with the organic solvents (ethanol, phenol, acetone, chloroform, and Isoamyl alcohol) (Fig. 7).
Effect of UV radiation on bacteriocin PB2
The influence of UV radiation on bacteriocin PB2 was studied. A similar pattern was observed against the reference strains with the time of exposure to the radiation. An indirect relationship was observed between the duration of exposure to UV radiation and the bacteriocidal activity of bacteriocin PB2 toward the reference strains. Bacteriocin PB2 demonstrated maximum bacteriocidal activity towards L. monocytogenes compared to E. coli (Fig. 8). Fig. 9 shows the purity and molecular weight of the amplicon from the probiotic strain PB2 which was later sequenced and submitted to Laragen Corporation [4]. A distinct band of 727 bp was observed (Fig. 9) from the PB2 strains (PB2a and PB2b).
Screening for the bacteriocin gene
After the plasmid isolation procedures of cured and uncured P. pentosaceus, amplification with primers of operon encoding pediocin PA-1, then visualization in a 1.2% (w/v) agarose gel stained with ethidium bromide was conducted. Afterward, the sample was run on the agarose gel along with the 1 Kb ladder. In the uncured P. pentosaceus strain (PB2+), plasmid screening revealed the presence of two plasmids with 0.93 and 1.2 kb sizes (Fig. 10). In the cured P. pentosaceus strain (PB2-), plasmid screening revealed that there were no plasmids (Fig. 10). Fig. 11 validates this result. Cured cells of Pediococcus pentosaceus (PB2-) didn't show any bacteriocin activity on the test strains, Listeria monocytogenes and Escherichia coli; while the uncured PB2+, displayed bacteriocin activity on the test strains.
Discussion
There is an increasing emergence of drug-resistant pathogens in the world. According to WHO [31], antimicrobial resistance (AMR) is one of the top ten global public health threats that humanity is facing. In Nigeria, AMR is compromising the treatment of infectious diseases like malaria, urinary tract infections, tuberculosis, digestive tract diseases including infectious diarrhea and inflammatory digestive tract diseases, and others. This is engendering an increased diversion from conventional medicine to traditional medicine. There are so many herbal and indigenous products that are being used and are effective against some of these diseases. A typical example is sorghum-fermented products, Pito and Ogi-baba which have been earlier reported to possess probiotic potentials against enteric-pathogenic diseases resulting from the presence of LAB [4]. This treatment option may be safer and cheaper compared to the high cost and potential toxicity associated with the use of chemical treatment.
LAB are found in fermented food like sorghum, meat, fish, potatoes, dairy products, pickles, fruits, humus, and the intestines of people and animals [32]. They are reported to possess antimicrobial properties [33]. Todorov et al. [13] reported that LAB like Lb. plantarum ST8SH has strong antimicrobial activity against L. monocytogenes ScottA, Lb. sakei ATCC 15521 and E. faecalis ATCC 19433 resulting from its bacteriocin.
Correspondingly, the results of this study revealed that P. pentosaceus PB2 (KP883297) isolated from Pito exhibits antagonistic activity against E. coli ATCC 25922 and L. monocytogenes ATCC 15313. This antagonistic property of P. pentasoceus PB2 defines its ability to inhibit the growth of Gram-positive and Gram-negative bacteria with a clear demonstration of bactericidal activity resulting from the acid production and bacteriocin effects. This is in agreement with reports of both Vizoso et al. [34] and Milette et al. [35] who independently deduced that the bactericidal effect of LAB could be attributed to its production of organic acids and bacteriocin-like proteins. Also, Ş imsek et al [36] and Ghosh et al. [10] had similar findings with Pediococcus sp. E5 and P. pentosaceus GS4 respectively. Hence, it can be inferred that the synergistic effect of lactic acid, diacetyl, hydrogen peroxide, and the bacteriocin produced by P. pentosaceus PB2 are responsible for antimicrobial activity.
This will be the first study to report the production of bacteriocin PB2, produced by P. pentosaceous PB2 isolated from Pito, a sorghum- based food in Nigeria. This study validates the claim that LAB from fermented sorghum products may show bacteriocidal activity resulting from antibacterial peptides [4]. Bacteriocin from the PB2 was purified and characterized using enzymology and biotechnology techniques [37]. A 2-step purification using 80% NH 4 (SO 4 ) 2 , and Carboxymethyl-Sephadex G-50 column chromatography was used to achieve a 12.62% purification fold and 13.3% yield of bacteriocin PB2. There are several reported protocols documented for bacteriocin purification some require multiple purification steps, while others do not [13,38,39]. Ladha and Jeevaratnam [39], then Vidhyasage and Jeevaratnam [40] likewise reported a high-fold purification.
Subsequently, the bacteriocin from the isolate PB2 was isolated, purified, and characterized and shown to have specific bacteriocidal activity of 2.64 U/mg with molecular weight (Mw) of 4.87 kDa. The molecular weight (Mw) of bacteriocins isolated from different sources varies, and they range approximately from 2700 to 16,599 Da [10,16]. The Mw shown by the bacteriocin ST8SH was estimated to be 5000 Da as reported by Todorov et al [13]. Similarly, a bacteriocin from Pediococcus pentosaceus CFR B19 had an Mw of ~4.8 kDa [41], and pediocin PD-1 from Pediococcus damnosus NCFB1832 had an Mw of ~2.6 kDa [42]. Bacteriocin PB2 is observed to have a larger molecular size when compared to bacteriocin PA-1 from Pediococcus pentosaceus strains [43], and pediocin PA-1 from P. acidilactici UVA1 [9]. Therefore, bacteriocin PB2 may be said to belong to class II of bacteriocins which are of small molecular weight < 10 kDa [12].
Physicochemical characterizations of bacteriocin PB2 showed stability at pH 3, 4, 5, 6, 7, 8, and pH 10, with the optimum pH of 5 while retaining activity after 2 h of incubation. Compared to the report by Todorov and Dicks [30] where the bacteriocin ST44AM was active at pH ranges 2 and 12, and Ladha and Jeevaratnam [39] where bacteriocin LJR1 was active at pH 4, 7, and 10, this study suggests that bacteriocin PB2 is active at a broad range of pH. The results show that PB2 can be stable at neutral, acidic, and basic pH.
Bacteriocin PB2 lost its antimicrobial activity when treated with proteinase K. This indicates the proteinaceous nature of the bacteriocidal agent [39,44,45]. The detergents were found to have no effects on the antimicrobial activity of bacteriocin PB2. Similar results to the detergent effect has been reported previously with bacteriocins ST44AM and LJR1 [39]. Also, the bacteriocin was found to be heat stable at temperatures 30, 40, 50, 60, and 80 • C for 2 h. Barathiraja et al. [46] highlighted that purified bacteriocin from the rumen liquor of a goat was unstable at 100 • C for 30 min, and stable at 30 • C. Bacteriocin PB2 was also observed to be stable upon the addition of organic solvents (ethanol, phenol, acetone, chloroform, and Isoamyl alcohol), surfactants, and detergents. This is in agreement with the findings of Kumar et al. [50] where they found bacteriocin LD4 from L. plantarum LD4 strain retaining activity after treatment with different organic solvents. Likewise, enterocin HDX-2 was treated with different organic solvents, surfactants, and detergents and was observed to be stable [26].
Exposure to UV light can alter the nature, structure, and function of a protein. Contrarily, bacteriocin PB2 retained its activity after six hours of exposure. Mahreen et al. [47] in their study observed that bacteriocin from L. helveticus retained activity following 30 min of UV exposure. Also, Shokri et al. [48] reported that BLIS from E. faecium DSH20 retained its activity after 30 min of exposure to UV light, the activity was retained. The result from this study shows that bacteriocin PB2 has better activity retention upon exposure to UV radiation.
In our study, isolation and amplification of genes encoding for the bactericidal activity of P. pentasoceus PB2 were performed targeting pediocin PA-1 genes in the total DNA. An amplicon of 727 bp was observed on agarose gel revealing its purity (Fig. 9), thereby indicating the production of a type of pediocin PA-1, which is a wide-spectrum bacteriocin from LAB. Murua et al. [49] used a similar method targeting plantaricin W, plantaricin S, plantaricin NC8 and pediocin PA-1 genes in L. plantarum LP08AD, and generated positive results only with the primers for plantaricin W, inferring that the organism is a plantaricin W producer.
Plasmid DNA preparations revealed two bands, from strain PB2, on agarose gel electrophoresis which are the two extrachromosomal DNA elements at 0.93 kb and 1.2 kb (Fig. 10) from the uncured plasmid strain PB2+. Similarly, Mathys et al. [9] associated the antimicrobial activity of Pediococcus acidilactici UVA1 with the presence of plasmids; they reported the presence of two plasmids at 9.5 kb and at > 10 kb, and found that the strain bac-lost its bacteriocidal activity after plasmid curing. In this present study, no extrachromosomal DNA was observed in strain PB2-, a non-inhibitory derivative of P. pentosaceus PB2 produced after plasmid curing. This was verified by conducting a plasmid curing screening on the agar (Fig. 11). The results infer that the plasmid encodes for the bacteriocin PB2 of P. pentosaceus PB2, hence, the bacteriocidal activity if the LAB.
Conclusion
Bacteriocin PB2, isolated from fermented sorghum product-Pito, was identified and characterized using a polyphasic approach. To our knowledge, Pediococcus pentosaceus PB2 is the first bacteriocinproducing Pediococcus strain isolated from fermented sorghum-based food in Nigeria, Pito. The results of the study show that P. pentosaceous exhibits antagonistic properties because of its identified bacteriocin. Some of the properties of the purified bacteriocin PB2 are a molecular weight of 4.87 kDa, an optimum temperature and pH of 40 • C, and pH of 5.0. Also, bacteriocin PB2 lost its activity on treatment with proteinase K and was observed to have stable activity in the presence of organic solvents. Remarkably, it retained its activity after six hours of exposure to UV light. P. pentosaceus was found to harbor two plasmids targeted from primers designed from operon encoding pediocin PA-1 gene whose molecular sizes of the plasmids are 0.9 and 1.2 kb. Biocomputational elucidation of the inhibitory potentials of bacteriocin PB2 against clinical protein targets of disease-causing microbes could be investigated for further insight into the mechanism of inhibition. This identified bacteriocin has great potential as an alternative treatment for drug-resistance strains of bacterial infections. This equally has the potential for biopreservation against food spoilage. More studies on the bacteriocin PB2 will have to be done for the aforementioned to be realized.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | 2022-10-31T15:10:55.816Z | 2022-10-01T00:00:00.000 | {
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222210213 | pes2o/s2orc | v3-fos-license | Benefits and barriers to pediatric tele-urology during the COVID-19 pandemic
Introduction Telemedicine video visits are an under-utilized form of delivering health care. However due to the COVID-19 pandemic, practices are rapidly adapting telemedicine for patient care. We describe our experience in rapidly introducing video visits in a tertiary academic pediatric urology practice, serving primarily rural patients during the COVID-19 pandemic. Objective The primary aim of this study was to assess visit success rate and identify barriers to completing video visits. The secondary aim identified types of pathologies feasible for video visits and travel time saved. We hypothesize socioeconomic status is a predictor of a successful visit. Materials and methods Data was prospectively collected and analyzed on video visits focusing on visit success, defined by satisfactory completion of the visit as assessed by the provider. Other variables collected included duration, video platform and technical problems. Retrospective data was collected via chart review and analyzed including demographics, insurance, and distance to care. Socioeconomic status was estimated using the Distressed Communities Index generated for patient zip code. Results/discussion Out of 116 attempted visits, 81% were successful. The top two reasons for failure were “no-show” (64%) and inability to connect (14%). Success versus failure of visit was similar for patient age (p = 0.23), sex (p = 0.42), type of visit (initial vs. established) (p = 0.51), and socioeconomic status (p = 0.39). After adjusting for race, socioeconomic status, and type of provider, having public insurance remained a significant predictor of failure (p = 0.017). Successful visits were conducted on multiple common pediatric urologic problems (excluding visits requiring palpation on exam), and video was sufficient for physical exams in most cases (Summary Table). A median of 2.25 h of travel time was saved. Conclusions While socioeconomic status, estimated using the Distressed Communities Index, did not predict success of video visits, patients with public insurance were more likely to have a failed video visit. There is compelling evidence that effective video visits for certain pathologies can be rapidly achieved in a pediatric urology practice with minimal preparation time.Summary Table Primary diagnoses addressed.Summary Table Visit Diagnosis N (%) Bladder and Bowel Dysfunction (dysuria, urgency, nocturnal enuresis, urinary incontinence, voiding dysfunction, or frequency with constipation) 35 (37%) Post-Operative Care 17 (18%) Penile Conditions (adhesions, hidden penis, phimosis, meatal stenosis, or hypospadias) 15 (16%) Prenatal Hydronephrosis (Postnatal visit) 11 (12%) Vesicoureteral Reflux 5 (5%) Nephrolithiasis 3 (3%) Prenatal Hydronephrosis (Fetal visit) 3 (3%) Scrotal Conditions (pain or swelling) 2 (2%) Vaginal Conditions (discharge or labial adhesions) 2 (2%) Ureteropelvic Junction Obstruction 1 (1%)
Introduction
Telemedicine video visits are an under-utilized form of delivering health care. However due to the COVID-19 pandemic, practices are rapidly adapting telemedicine for patient care. We describe our experience in rapidly introducing video visits in a tertiary academic pediatric urology practice, serving primarily rural patients during the COVID-19 pandemic.
Objective
The primary aim of this study was to assess visit success rate and identify barriers to completing video visits. The secondary aim identified types of pathologies feasible for video visits and travel time saved. We hypothesize socioeconomic status is a predictor of a successful visit.
Materials and methods
Data was prospectively collected and analyzed on video visits focusing on visit success, defined by satisfactory completion of the visit as assessed by the provider. Other variables collected included duration, video platform and technical problems. Retrospective data was collected via chart review and analyzed including demographics, insurance, and distance to care. Socioeconomic status was estimated using the Distressed Communities Index generated for patient zip code.
Results/discussion
Out of 116 attempted visits, 81% were successful. The top two reasons for failure were "no-show" (64%) and inability to connect (14%). Success versus failure of visit was similar for patient age (p Z 0.23), sex (p Z 0.42), type of visit (initial vs. established) (p Z 0.51), and socioeconomic status (p Z 0.39). After adjusting for race, socioeconomic status, and type of provider, having public insurance remained a significant predictor of failure (p Z 0.017). Successful visits were conducted on multiple common pediatric urologic problems (excluding visits requiring palpation on exam), and video was sufficient for physical exams in most cases (Summary Table). A median of 2.25 h of travel time was saved.
Conclusions
While socioeconomic status, estimated using the Distressed Communities Index, did not predict success of video visits, patients with public insurance were more likely to have a failed video visit. There is compelling evidence that effective video visits for certain pathologies can be rapidly achieved in a pediatric urology practice with minimal preparation time.
Introduction
The COVID-19 pandemic has had an enormous impact on pediatric urologic practices throughout the world, necessitating postponement of elective surgical procedures and non-urgent patient appointments [1,2]. Emergency surgeries continue to be performed, but questions remain as to what non-urgent surgeries can safely be postponed [2,3]. Similar questions arise for postponement of office visits. Delay of pediatric urologic care in certain cases (e.g. obstructive uropathy) could lead to worse outcomes or allow for progression of irreversible conditions, without being immediately life-threatening. To prevent morbidity associated with the postponement of care during the COVID-19 pandemic, methods that safely allow for high quality care have been implemented. The COVID-19 pandemic has drastically accelerated the adaptation of telemedicine within pediatric urology [4]. Telemedicine allows for the delivery of care while maintaining safe social-distancing measures. Remote video visits (VVs) have been used successfully for years in pediatric urology for the delivery of postoperative care [5,6] and prenatal urologic consultation [7]. Yet overall, there is a paucity of existing research on the use of telemedicine for general pediatric urologic conditions.
The University of Virginia (UVA) is an academic tertiary care center catering to a mainly rural population across the state of Virginia. Beginning in March 2020 in response to COVID-19, the pediatric urology department at UVA began offering VVs to patients for the first time. This study seeks to present our experience of rapidly integrating the use of VVs into a previously in-person only practice. The primary aim was to assess the rate of successful VVs and identify barriers to a successful remote VV. The secondary aim was to delineate what types of visits were more feasible for VVs and how much travel time was saved by VVs.
Material and methods
Institutional Review Board approval was obtained (IRB #22340). In March 2020, the Pediatric Urology Division began rescheduling non-urgent patient appointments to telephone visits, VVs, or postponed in-person visits. Four providers (2 pediatric urologists and 2 nurse practitioners) selected the visits that were deemed appropriate for VV and conducted all of the VVs. Visits excluded from VV included conditions that required palpation on physical exam (e.g. undescended testis) and visits that required imaging (e.g. ultrasound, fluoroscopic imaging, etc.) that was unable to be obtained due to COVID-19. VV platforms (doxy.me and Facetime) were selected based on institutional approval and patient preference; during the initial pandemic-related clinic closures, Facetime was given institutional approval for telemedicine use. Patients did not receive pre-clinic check-ins or phone calls to obtain initial intake. Data was prospectively collected on patients scheduled for a VV from March 27, 2020 to May 15, 2020. Providers collected data on visit outcome (success vs. failure), duration, VV platform, and any technical problems encountered. VV success was defined as satisfactory completion of the clinical visit using VV technology as assessed by the provider. Conversion of a VV to telephone appointment due to technical difficulties or a "no-show" was considered a failure. Patients were present with their parent/guardian at all VVs.
Following the VV, retrospective data were collected by chart review. Patient data collected included age, race, zip code, and type of insurance (public, private, or no insurance). VV data included primary diagnosis as well as the clinical outcome of the visit (e.g. medication change, imaging studies, surgery). The patient's originally scheduled appointment location, cancelled due to COVID-19, was noted (main campus or satellite clinic). Roughly 15% of patients are typically seen at one of the 4 satellite clinics located between 30 and 118 miles away from the main campus. The distance from their home zip code to the original appointment location was then entered into Google Mapsä to estimate the travel distance and time they saved, using traffic conditions for 1:00 PM on a Wednesday. When a patient had no original visit, it was assumed they would have been scheduled for an appointment at the main campus location. The average distance of the shortest route and time to the clinic were recorded. Roundtrip time was estimated by doubling these values.
Socioeconomic status (SES) was estimated using the Distressed Communities Index (DCI) generated for each patient's zip code. Created by the Economic Innovations Group, the DCI was generated using the U.S. Census Bureau's American Community Survey's 5-Year Estimates and Business Pattern data from 2007 to 2011 and 2012e2016 [8]. The DCI generates a normalized, comparative distress score ranging from 0 ("prosperous") to 100 ("distressed") for each zip code. The distress score is a composite of seven variables: rate of graduation, housing vacancy rate, unemployed adults, poverty rate, median income, change in employment, and change in the number of business establishments. DCI scores were licensed for use in this publication.
Statistical analysis
Descriptive variables are presented as median (interquartile range IQR) and count (percentage) as appropriate. Independent t-tests and Chi-square tests were used as appropriate to compare successful versus failed VV based on characteristics described above. Univariate and multivariable logic regression modeling was used to analyze the effect of study variables on VV success. The final model included patient and clinical variables with p < 0.20 on univariate analysis. Study data were collected and managed using REDCap electronic data capture tools [9]. SAS version 9.4 (SAS Institute Inc. Cary, NC) was used to perform statistical analysis with a statistical significance threshold set at 0.05.
Primary outcome
During the seven-week study period, 116 patients scheduled for a VV were included in our cohort. Three additional patients were asked to participate in a VV but declined due to lack of internet availability. Of the 116 VV attempts, 94 were successful and 22 failed for an overall VV success rate of 81%. Of these patients, 5 initially had a failed visit due to "no-show" but later had a successful visit. Otherwise, no patients in our sample had multiple VVs. Median length of successful VV was 22 min (IQR 15e30). Clinical outcomes of these 94 visits resulted in further testing with imaging or labs (37.2%), prescription changes (21.2%), surgery/procedure scheduling (10.6%), and follow-up visit only (46.8%). Reasons for VV failure included "no-show" (63.6%) and inability to connect (13.6%). For all patients who were unable to connect, the VV was converted to a telephone visit. The remaining 22.7% failed for several reasons: one patient's parent required an interpreter, one patient's parent was not sent the web browser link for the appointment, one patient's parent requested to reschedule the visit, one patient's parent decided to proceed with a telephone visit instead without video, and in one case, another child in the household was using the computer for school during the appointment (Table 1).
Doxy.me was the most commonly used video platform attempted (84%). Patients who successfully completed a VV were similar to those who failed in age (p Z 0.23), sex (p Z 0.42), type of visit (initial vs. established) (p Z 0.51), and socioeconomic status as estimated by median DCI score (p [ 0.29) ( After adjusting for race, socioeconomic status, and type of provider, having public insurance (vs. commercial insurance or no insurance) remained a significant predictor of VV failure [OR, 3.85; 95% CI 1.28e11.60; p Z 0.017] ( Table 3).
Secondary outcome
The most common primary diagnoses addressed for a VV included bladder and bowel dysfunction (37%), post-operative care (18%), and penile conditions (16%) ( Table 4). Most visits were for established patients (62%) and less for new patients (38%). 33% of patients had imaging done prior that was discussed during their VV. All imaging was reviewed by the provider without the use of outside reports. The majority of VVs that required a physical exam was completed successfully. Out of 37 initial visits, 14 required a physical exam to determine the plan of care. Eight exams were successfully performed via video for diagnoses including penile adhesions, hypospadias, labial adhesions and circumcision/circumcision revision. Six exams were considered difficult. For 3 patients, difficulties were due to patient movement and grainy picture quality to evaluate the conditions including penile skin bridging, hypospadias/chordee, and meatal stenosis. Three additional patients required in-person testicular exams for palpation purposes to evaluate scrotal pain or undescended testis. Of note, the undescended testis was not the primary reason for the visit but was incidentally discussed by the parent. For the patients with inadequate video exams, inperson clinic follow-up was requested. Out of 57 followup and post-operative VVs, 19 required physical exams to evaluate post-operative healing or determine future plan of care. All of these exams were adequate via the video platform. Post-operative VVs were performed for hypospadias repair, orchidopexy/orchiectomy, inguinal hernia repair, circumcision, and chordee repair.
Patients who successfully completed a VV would have travelled a median 101.4 miles (IQR 51.4e155.9) roundtrip with a median travel time of 135 min (IQR 72.9e200) to get to the in-person appointment.
Discussion
Prior to the COVID-19 pandemic, telemedicine was an under-utilized form of delivering health care in the field of pediatric urology [10]. Prior studies have demonstrated successful telemedicine encounters for post-operative visits as well as for prenatal consultations on urologic conditions, most commonly including urinary tract dilation [5e7]. Out of necessity, the pandemic has changed the way practitioners have traditionally provided services in order to maintain quality care for pediatric urologic patients. Due to this unforeseen adaptation of telemedicine into a solely in-person practice, this study aimed to understand factors that may lead to a successful VV and also understand barriers to the process. VVs commenced within one week of hospital wide changes in response to COVID-19. Despite the minimal preparation time, our VVs were largely successful at an 81% rate. In scheduled visits excluding no-shows, our technical success rate was 92%. A previous pediatric urology telemedicine investigation by Finkelstein et al. found a similar rate of 96% [6]. No patients in the study had previous telemedicine visits with our practice, so the technical success rate did not benefit from patient experience with previous pediatric urology telemedicine visits. We still had a modest no-show rate, totaling 12%; this rate was fairly surprising given the state had a stay-at-home order at the time. This rate was certainly higher than the rate reported by Finkelstein et al., at 6% [6]. There are many possible reasons for this discrepancy including demographics, nature of the visits, and potential differences in scheduling and reminders. While post-operative visits comprised 18% of our cohort, Finkelstein's study included only post-operative visits [6]. Postoperative visits have previously been found to be better attended than other follow-up appointments in an otolaryngology study [11] that may be explained by patients investing more in an appointment after a surgery. Only some of the patients received pre-appointment reminders, and these reminders are a proven method to reduce no-show rates for in-person visits [12]. Additionally, our success rate could have been higher if we included telephone conversion as a successful visit given many providers may feel telephone is sufficient. However, we did not consider these converted visits as a success given the element of video or visualization of the patient can be an important component of an office visit.
In the course of this study, we sought to understand what factors predict a failed VV. We presumed patient age and sex would not be predicting factors given the parent or guardian was the participant in the VV and not the child. We unfortunately did not have data on parents' demographics. We had anticipated higher SES would be positively correlated with visit success, as bandwidth and SES have been found to be barriers to the use of telemedicine in the past [13]. Alternatively, we found that DCI, a proxy for SES, did not predict visit failure. The DCI has been used in several medical studies including recent publications examining the relationship between SES and surgical risk [14,15]. We, however, recognize the flaws in using DCI as a surrogate for SES given this score generalizes a community to their zip code and does not account for individuals who deviate within the mean. Additionally, given the popularity and wide-spread use of smart phones and computer technology in 2020, failure of VVs may not be representative of low SES as it may have been in the past. Interestingly, public insurance, that may be considered another alternate for low SES, significantly predicted a failed VV in a multivariate analysis.
Beyond the feasibility of VVs, there are many advantages of telemedicine for patients and families including a reduction in pre-appointment wait time [9,14], greatlyreduced travel time and rate of missing school or work for the appointment with equivalent clinical outcomes as compared to in-person visits [16]. Obviating the need to travel is an important benefit to emphasize, as our patients live throughout the state of Virginia and often need to travel great distances to attend an appointment. The patients in our cohort would have saved a median 2.25 h in travel time. Although we serve a mainly rural population, the experience of our patients' families is not unique, as it has been found that 29% of the under-18 population in the United States lives at least 40 miles from the nearest pediatric urology practice [17].
This time and travel savings has to be balanced with providing good quality care remotely. Within our cohort, the VVs were productive in that they lead to a change in plan of action in 69% percent of cases (i.e. prescription change, surgery or imaging scheduling, etc). To our knowledge, this is the first study to demonstrate the utility of telemedicine in pediatric urology beyond prenatal consultation and postoperative visits. In our study, telemedicine was used for decision-making that led to changes in care including surgery and medication changes. In scheduling the patients for VV, we recognized that a large number of patients required imaging results to have a meaningful visit. If patients were unable to get these studies, their visits were required to be delayed and did not merit a VV. Another barrier with telemedicine is the lack of ability to perform a thorough physical exam, especially one that requires palpation. Therefore, patients with undescended testes were strategically deferred for an inperson visit. Other conditions such as identifying a urethrocutaneous fistula or meatal stenosis after hypospadias repair may be quite challenging. From our secondary outcome, we found that post-operative visits were quite amenable to VV as previously demonstrated by others [6], with no difficulties in examination; however 43% of exams were considered inadequate on initial visits that were reliant on the physical exam. While the number of difficult exams was small, it highlights that certain conditions are better suited for telemedicine compared to others. Future research should focus on success rates and outcomes of VV appointments broken down by diagnosis.
Several limitations should be noted. Our sample is a largely self-selected group of patients that had access to the equipment necessary for VVs, therefore may hold selection bias. It would be prudent to examine the rates of patients turning down the opportunity for VVs to gain a better understanding of what barriers in the community prevent patients from even attempting a VV. We also did not have demographic data on the parents to help identify factors that could influence the success or failure of VVs. Additionally, although providers deemed visits successful, it would be beneficial to examine patients' perception on whether they feel their goals were accomplished. Equally so, a comparison between VVs and in-person visits may be informative. Finally, as mentioned above, although we used DCI as a surrogate for SES, this score is certainly flawed due to its generalization based on zip code.
Conclusions
Our results provide compelling evidence that effective remote VVs can be rapidly achieved in a pediatric urology practice with minimal preparation time. Private insurance status was the most predictive factor for a successful VV. | 2020-10-09T13:06:56.713Z | 2020-10-08T00:00:00.000 | {
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269147597 | pes2o/s2orc | v3-fos-license | White matter lesion load and location in relation to cognitive impairment in relapsing– remitting multiple sclerosis
Background In relapsing–remitting multiple sclerosis (RRMS) the connection between cognitive impairment (CI) and white matter lesion load (WM-LL) and location is still unclear. This study aimed to identify the relationship between CI in RRMS patients and WM-LL and locations using a fully automated platform. CI and WM-LL were evaluated in 90 patients with RRMS using the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) and Automated MRI volumetric measures of WM-LL and lesion distribution. Regression analysis of BICAMS as a dependent variable with different clinical and radiological parameters was performed. Results Data were obtained from 90 patients with RRMS who had a mean age of 32.74 ± 8.43 years and a female-to-male ratio of 3:1. The mean (± SD) cognitive rating scores for the BICAMS subtests were 28.07 ± 11.78 for the Symbol Digit Modalities Test (SDMT), 42.32 ± 12.46 for the California Verbal Learning Test-II (CVLT-II), and 16.13 ± 8.17 for the Brief Visuospatial Memory Test-Revised (BVMT-R). According to the BICAMS criteria, 29 cases (32.2%) had CI. BICAMS scores were significantly correlated with age, education level, relapse frequency, disease duration, and time to start disease-modifying therapies. Whole WM-LL and periventricular lesion load were significantly associated with CI. After controlling for age, sex, and education, logistic regression analysis revealed that total WM-LL was the best predictor for CI together with duration of illness and years of education. The cut-off value of 12.85 cc for total WM-LL predicted CI. Conclusions Whole WM-LL and periventricular lesion load are the best anatomical predictors for CI probably due to the effect on the anterior commissural fibers while years of education and duration of disease are the best demographic predictors for CI.
Background
Multiple sclerosis (MS) is the most common cause of non-traumatic disability among highly productive young and middle-aged adults [1].Even though physical disability is the hallmark of the disease, cognitive impairment (CI) has also been recognized as a central feature, affecting up to 70% of patients [2].CI in the context of MS is evident even at disease onset and increases in both prevalence and severity as the disease progresses [3].
CI can result in difficulties in employment, treatment non-adherence, personality changes, and other psychosocial dysfunctions in patients and even their careers [4].Therefore, it is essential for health professionals to evaluate cognitive function objectively both at baseline and during routine follow-up visits [5].
MS-related cognitive deficits mirror subcortical dementia, with effects on attention, information processing speed, memory, executive function, and visuospatial abilities [6].The Symbol Digit Modalities Test (SDMT), the California Verbal Learning Test-II (CVLT-II), and the Brief Visuospatial Memory Test-Revised (BVMT-R) are subtests of the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS), a popular assessment battery that has strong associations with numerous MRI measures in MS patients [7].The widespread use of BICAMS stems from its short administration time (only 15 min), multilingual accessibility, and high sensitivity and specificity in evaluating information processing speed and short-term verbal and visual memory [7].
There is some debate over whether MRI volumetric assessments of white matter (WM) and grey matter (GM) structures can be used as predictors of CI and quantify a patient's response to treatment [8][9][10].It is possible that discrepancies in the literature may relate to diverse patient profiles, insufficient numbers, different rating scales for assessment, various definitions of CI, different lesion quantification methods, or failure to take lesion location into account [11].The present study aimed to evaluate the relationship between CI, as measured by BICAMS and physical disability, as measured by EDSS, with different automated MRI measures of white matter lesion load (WM-LL) (lesion volume, normalized volume, lesion burden), lesion distribution and other volumetric assessments.
Participants
RRMS patients were recruited consecutively from three MS units located in the South of Egypt (Assiut, Sout Valley and Luxor) during the period from the 1st of January to the end of September 2023.Patients were diagnosed with RRMS according to the 2017 McDonald diagnostic criteria [12].Inclusion criteria: RRMS patients of all ages and either sex, both de novo and old cases attending the MS units during the study period.Exclusion criteria: (a) any patient with evidence of a relapse or steroid administration in the past 30 days; (b) any associated medical conditions that may affect cognition, and those on psychoactive pharmacotherapy; (c) patients who were unable to complete the test as due to visual impairment, upper limb weakness or tremors were also excluded; (d) patients with incomplete clinical data or could not complete all cognitive assessments; and (e) patients without MRI image DICOM files (see flowchart Fig. 1).
Study procedures
This was a cross-sectional, each patient was submitted to the following.
Clinical evaluation
Demographic data (age, sex, educational level represented by the number of educational years) were provided, followed by clinical history and neurological examination including the Expanded Disability Status Scale (EDSS).
Cognitive assessment
Cognition was assessed using the validated Arabic version of BICAMS battery [13].The BICAMS battery includes SDMT for evaluating the speed of information processing, CVLT-II for assessing verbal learning and memory, and BVMT for evaluating visual learning and memory.The tests were applied in quiet rooms in the MS outpatient clinics on the morning of the scheduled visit just before acquisition of the MRI.The tests were applied by 3 neurologists (one neurologist in each center) who attended a training session on the application of BICAMS before the start of the study to decrease any potential inter-rater variability.Scoring of different subtests was done by the same neurologist.Application and scoring of the three subtests were done according to the international recommendations [14].A group of age, sex and education-matched control group was used to determine the cut-off values for SDMT, CVLT-II, and BVMTR (22, 38, and 10, respectively).These values were calculated as 1.5 SD below the mean of the healthy age, sex, and education-matched group [15].Patients were classified as having CI if at least two tests were abnormal [14].
Lesion load and volumetric assessment
The volumetric data were acquired from MRI data that included T1 and FLAIR sequences.These sequences were originally converted from DICOM files to two compressed, anonymized Neuroimaging Informatics Technology Initiative (NIfTI) files: one for T1 and one for FLAIR.These NIfTI files formed the foundation for further analysis.
LesionBrain 1.0 is a web-based program for segmenting white matter lesions [16] that was successfully incorporated into the platform "volBrain" (https:// volbr ain.upv.es/) [17], offering a complete tool for precise and efficient lesion detection.Image normalization and registration, structural segmentation to identify particular brain areas such as the intracranial cavity, brainstem, cerebellum, and lateral ventricles, and candidate mapping to locate probable lesion sites are all part of the processing pipeline.Following that, lesions are segmented voxel-wise using a three-step technique that includes patch-based multimodal segmentation, patch-based regularization of the resultant lesion probability map, and an ensemble of shallow neural networks to correct any erroneous patches, decreasing false positives.This pipeline has been rigorously tested using the MSSEG MICCAI Challenge 2016 dataset, displaying good performance with a mean Dice coefficient of 0.66 [18].This thorough technique enabled the absolute volume of all lesions, whether total or localized, to be measured (in cubic centimeters).
Statistical analysis of data
SPSS for Windows version 26 was used to perform statistical analysis (Cary, NC, USA).Data were represented as mean and standard deviation for numerical data and frequencies with percentages for categorical data.The Shapiro-Wilk and Kolmogorov-Smirnov tests were employed to determine the normality of the distribution.
To assess the differences and correlations between variables, suitable parametric or nonparametric tests were utilized.Pearson or Spearman correlation tests were used to determine the relationship between variables.To evaluate the link between variables, multivariate logistic regression and linear regression analysis were used.For all tests, a P-value of less than 0.05 was considered significant.
Clinical data and brain volumetric measures
Data were obtained from 90 patients with RRMS who had a mean age of 32.74 ± 8.43 years and a female-tomale ratio of 3:1.The mean (± SD) of EDSS was 3. 1.
Comparisons between patients with and without CI are illustrated in Table 2. Patients with CI were significantly older, had fewer years of education, and had a longer duration of illness with a higher number of relapses.They also had higher EDSS scores, total WM lesion volumes, and Table 3 illustrates the results of the correlation analyses.Age, years of education, disease duration, number of relapses, EDSS, DMT duration, and time to start DMT were significantly correlated with all subtests of the BICAMS, while no correlation was found with age at onset of illness.Total WM-LL and periventricular lesion volume showed significant negative correlations with all BICAMS subtests.
The results of multivariate stepwise logistic regression analysis, with adjustment for age, sex, and current DMT are illustrated in Table 4.The key variables were years of education (inverse relationship with cognitive impairment, P = 0.008), disease duration (positive association, P = 0.004), and WM-LL (positive association, P = 0.042).These findings highlight the significance of education years, disease duration, and lesion volume as predictors of CI.
Table 5(A) and Fig. 2 show the ROC curve and an area under the curve (AUC) of 0.700 for total white matter lesion load (Total WM-LL) in diagnosing cognitive impairment (CI).The AUC, with a standard error of 0.057, is statistically significant (P = 0.002), suggesting moderate predictive accuracy.
Table 5(B) provides a cut-off value of 12.85 cc for total WM-LL, yielding a sensitivity of 75.9% and specificity of 60.7%.
Discussion
Although MRI parameters of structural brain damage are closely linked to cognitive disabilities, they are not incorporated in routine clinical assessment due to the need to employ sophisticated types of analysis and measurements of volumetric MRI measurements which are not routinely available in most centers [19].The reliability of these measures using largely automated approaches has not been proven, while studies employing other methods yielded conflicting results [20].
In the present study, CI was found in 32.2% of patients which is lower than the frequency of CI reported in several previous studies which ranged from 40 to 70% [21].Compared with patients who had normal cognition, patients with CI were older, had fewer years of education, a longer duration of illness with higher relapse frequency, a higher EDSS, and higher total WM-LL and periventricular LL.Each of these items was also significantly correlated with the three subitems of BICAMS.Similar results have been reported by Khedr and colleagues, 2022, and Elshebawy and colleagues 2021 [15,22].Many reports have pointed out that increased age is a risk factor for cognitive decline in MS [15,[22][23][24].Aging is usually associated with brain atrophy and CI in the general population while in MS, this rate of atrophy is accelerated [25,26].Aging increases the probability of secondary progression in MS due to exhaustion of brain reserve [27].Cognitively impaired patients had higher EDSS scores [28, 29].Since physical performance requires higher-order information processing, understanding the relationship between cognition and physical performance is important when considering cognition as an important risk assessment measure [30].The only protective factor for physical disability was a higher educational level due to increased cognitive reserve and brain plasticity [31].
The findings of the present study suggest that there may be differences in cognitive performance among MS patients according to the type of disease-modifying therapy (DMT).Patients receiving Interferon B had higher scores on visuospatial memory as measured by the Brief Visuospatial Memory Test-Revised Total Recall (BVM-RT), despite their lower level of education.Although they had similar EDSS scores and duration of illness, they also had a significantly lower relapse rate than patients receiving fingolimod as the latter is usually considered as 2nd line of treatment in such cases.
The main results of the present study are that three factors can be considered as predictors of CI in RRMS: years of education, disease duration, and whole WM-LL.
Education years
The odds of cognitive impairment decrease for each additional year of education.This is consistent with Elshebawy and colleagues, and Khedr and colleagues, who found that a low educational level was a predictor of CI in MS patients [15,22].Education is one of the factors responsible for the formation of cognitive reserve.A higher educational level is thought to increase the brain's resilience to disease burden, at least up to a certain limit [32].Several studies have linked better cognitive performance with higher education levels and cognitive reserve [15,22,33,34].However, Russo and colleagues [35] and Patti and colleagues [36] found no significant differences between cognitively preserved or impaired patients regarding their educational level.
Disease duration
We found that for each additional year of disease duration, the odds of CI increase.This aligns with the studies above, which identified longer disease duration as a predictor of CI [22].
Lesion volume
We found that for each unit increase in lesion volume, the odds of cognitive impairment increase.The area under the curve of total WM-LL predicting CI was 0.700 (95% confidence interval 0.589-0.810).The optimal cutoff value of the total WM-LL predicting CI was equal to or greater than 12.85 cc with a sensitivity of 75.9% and specificity of 60.7%.Calculation of the cut-off value of WM-LL could be utilized for early detection of CI even Fig. 2 Shows ROC curve for total white matter lesion load (WM-LL) for diagnosing cognitive impairment.The ROC curve and an area under the curve (AUC) of 0.700 for Total WM-LL in diagnosing cognitive impairment (CI).The AUC, with a standard error of 0.057, is statistically significant (P = 0.002), suggesting moderate predictive accuracy in asymptomatic patients.However, it is important to note that cognitive impairment in MS is a complex issue and can be influenced by many factors such as frequent relapses, progressive form, higher clinical disability, and immunosuppressive treatment [22].It might be beneficial to consider these additional factors in future analyses.
Although most previous studies agree that there is a relationship between WM-LL and CI, as reflected in the statements of the National MS Society [37].Fulton and colleagues discovered that out of 12 neurocognitive indicators assessed, only SDMT and Rey Auditory Verbal Learning test were associated substantially with lesion burden [38].Patti and colleagues (2015) observed that aberrant white matter (AWM) percentage, a marker of lesion burden, predicted poor performance in SDMT Test 9 years in this study [39].According to Giorgio et al., the connection between CI and lesion burden is modest, suggesting that CI in MS has a complicated and multifaceted etiology that is insufficiently described by pathological markers detected by standard MRI [40].Nocentini et al. demonstrated that CI was linked with global brain atrophy and T2-lesion volumes as determined by voxelbased morphometry (VBM) [41].Another research employing 3.0 T MRI with enhanced identification of tiny lesions undetected by conventional MRI found a significant connection between CI and WM-LL [42].A recent Egyptian study that compared WM-LL in double inversion recovery (DIR) with SDMT cognitive scores discovered that the total WM-LL was adversely connected with SDMT cognitive scores [43].
Overall, the connection between cognitive performance and lesion burden is modest, suggesting that cognitive impairment in MS has a complex and multifaceted etiology that is not fully described by pathological markers detected by standard MRI [40].While some studies have found a significant correlation between regional lesion load and CI in MS patients, others suggest that this relationship is moderate or complex.More research is needed to fully understand this relationship.
Disruption of cortico-cortical and cortico-subcortical connections involved in cognitive processing may be the mechanism through which WM-LL causes deficiencies in specific domains of cognition [39].According to Reuter and colleagues, CI is present in approximately one-quarter of MS patients in the early stages, and the location of macroscopic lesions altered performance in verbal and spatial learning but had no effect on attention and executive functioning [44].Despite the comparable anatomical distribution of WM-LL, Rossi and colleagues discovered that lesion volume was larger in cognitively impaired individuals than in cognitively preserved patients [45].Khedr and colleagues, 2023 found that GM atrophy particularly thalamic atrophy was the best predictor of CI as they measure only the gray matter in their study [15].Kutzelnigg and colleagues showed that, with disease progression, WM abnormalities become more diffuse with increased demyelination in the GM [46].Since neither WM nor GM abnormalities alone could fully explain CI in MS, WM lesion location may give a stronger correlation with the severity of cognitive impairment [47].It has been shown that lesions in WM tracts connecting associative areas are correlated with CI in RRMS patients [48,49].The effect of WM-LL in strategic tracts in predicting CI may be far more important than the proposed diffuse abnormalities in the normal-appearing WM [50].
Confirming our result Papadopoulou et al. found that WM lesion volume significantly predicted SDMT and by trend PASAT performance [51].However, cortical lesion volume did not predict CI.Others [52,53] [15,54,55].Integration of the relatively simple measures of automated brain volumetry and WM-LL and location in routine monitoring of RRMS patients may be able to detect patients' early patients with CI and consequently could be detecting transiting to secondary progression and allow early intervention.Recently Kania et al. concluded that baseline volumetric measures are stronger predictors of cognitive performance than relapse activity, which yet again highlights the importance of atrophy in MS prognosis [56].
The present study was cross-sectional and did not allow us to shed light on the longitudinal changes in the relationship between the quantitative MRI measures of WM-LL with CI over the course of the disease.Also, the use of a 1.5 T MRI scanner did not allow more advanced MRI measurements (WM tractography).We recommend further studies on larger populations and on other phenotypes like PPMS for a better understanding of the underlying correlates.
Conclusion: In the current study, total WM-LL can be used as a predictor of CI and this finding means that the CI in RRMS is a subcortical type of impairment due to periventricular WM-LL.It may be related to affection of anterior commissural fibers of corpus callosum while years of education and duration of disease are the best demographic predictors for CI.
Fig. 1
Fig. 1 Flowchart shows the inclusion and exclusion of the study participants RRMS Patients recruited consecutively from 1st of January to the end of
Table 1
The demographic and clinical data of 90 RRMS patients DMT disease-modifying therapy, SDMT Symbol Digit Modalities Test, CVLT-II California Verbal Learning Test-II, BVMT-R Brief Visuospatial Memory Test-Revised, EDSS Expanded Disability Status Scale, WM-LL white matter lesion load
Table 2
Comparison between patients with and without cognitive impairmentDMT disease-modifying therapy, EDSS Expanded Disability Status Scale, WM-LL white matter lesion load
Table 3
Correlation of different sub-items of BICAMS to study variables * Statistically significant difference (p < 0.05) **Statistically significant difference (p < 0.01) DMT disease-modifying therapy, EDSS Expanded Disability Status Scale, WM-LL white matter lesion load
Table 4
Multivariate stepwise logistic regression analysis for predicting CI using clinical and volumetric results, with adjustment for age, sex and current DMT administered
Table 5
A. Area under the curve and B. Cut-off value of total WM-LL for diagnosing cognitive impairment A.
-off value Positive if greater than or equal to Sensitivity Specificity
found the cortical lesions and atrophy associated with cognitive impairment in RRMS while Akaishi et al., and Naghavi et al., and Khedr et al. found that deep gray matter atrophy is highly correlated with overall cognitive impairment in RRMS.This controversy in results may be related to methodological differenced in measuring MRI | 2024-04-16T13:17:22.379Z | 2024-04-15T00:00:00.000 | {
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270407143 | pes2o/s2orc | v3-fos-license | The Impact of the COVID-19 Pandemic on Incidence and Short-Term Survival for Common Solid Tumours in the United Kingdom: A Cohort Analysis
Purpose The COVID-19 pandemic profoundly affected healthcare systems and patients. There is a need to comprehend the collateral effects of the pandemic on non-communicable diseases. We examined the impact of the pandemic on short-term survival for common solid tumours, including breast, colorectal, head and neck, liver, lung, oesophageal, pancreatic, prostate, and stomach cancer in the UK. Methods This was a population-based cohort study of electronic health records from the UK primary care Clinical Practice Research Datalink GOLD database. In sum, 12,259,744 eligible patients aged ≥18 years with ≥1 year’s history identified from January 2000 to December 2022 were included. We estimated age-standardised incidence and short-term (one- and two-year) survival for several common cancers from 2000 to 2019 (in five-year strata) and compared these to 2020–2022 using the Kaplan–Meier method. Results Incidence decreased for most cancers in 2020 and recovered to different extents in 2021–2022. Short-term survival improved for most cancers between 2000 and 2019, but then declined, albeit minimally, for those diagnosed in 2020–2022. This was most pronounced for colorectal cancer, with one-year survival falling from 78.8% (95% CI 78%–79.6%) in 2015–2019 to 77% (95% CI 75.6–78.3%) for those diagnosed in 2020–2022. Conclusion Short-term survival for many cancers was impacted, albeit minimally, by the pandemic in the UK, with reductions in survivorship from colorectal cancer equivalent to returning to the mortality seen in the first decade of the 2000s. While data on longer-term survival are needed to fully comprehend the impact of COVID-19 on cancer care, our findings illustrate the need for an urgent and substantial commitment from the UK National Health Service to address the existing backlog in cancer screening and diagnostic procedures to improve cancer care and mortality.
Introduction
The COVID-19 pandemic left a permanent mark on global public health, with profound implications for healthcare systems and patients.Whilst healthcare systems grappled with the overwhelming demands of treating COVID-19 patients, there emerged a pressing need to comprehend the collateral effects of the pandemic on non-communicable diseases, including cancer.2][3] Reduced screening and diagnostic tests inevitably cause delays, leading to more advanced stage at diagnosis and worsened prognosis.
Survival from cancer has generally improved over the last two decades, with combined mortality rates across all cancers and sexes decreasing by around 10%. 4 One study estimated that in the UK, the probability of dying from cancer before 80 years of age declined from 0.16 to 0.13 for women and 0.22 to 0.17 for men across 2002-2019. 5The improvement in cancer survival can be attributed to several interconnected factors that have contributed to advancements in cancer prevention: screening, early diagnosis, and treatment. 6These factors have collectively led to better outcomes and increased survival rates for cancer patients.However, the pandemic's disruptive impact on healthcare systems and cancer care delivery has raised concerns about delayed diagnosis, altered treatment regimens, delays in surgery, and reduced access to essential services, all of which may have adverse consequences for cancer patients' survival.
The UK's National Health Service (NHS) was already under pressure prior to the pandemic, suffering from years of reduced funding and substantial challenges, including staff shortages and reduced hospital beds per capita. 7Staffing issues were further compounded because many doctors and nurses were on sick leave or isolating due to COVID-19 exposure.Surgical staff were redeployed to care for COVID-19 patients, and operating theatres and outpatient clinics were closed to free up resources for patients with COVID-19. 8This led to the halting of routine and elective care in the months following the outbreak. 9Despite these efforts, the rapid spread of COVID-19 in the UK impacted health and social care nationally, and the government implemented three national lockdowns.
Literature on the impact of the pandemic on survival from cancers is currently limited, with only a handful of studies addressing this in the UK. 10,11Given the prioritisation of urgent care for COVID-19 during the pandemic on top of a stretched NHS, we hypothesised that delays in the diagnosis and/or lack of cancer screening would lead to increased short-term mortality due to more advanced stages at diagnosis.We speculated that the expected delay in diagnosis and hence potentially reduced short-term survival would be more noticeable for cancers that benefit from early detection and for which we have successful screening programmes (such as breast and colorectal cancer).
The overall aim of this study was to investigate the impact of the COVID-19 pandemic and its management on short-term (one and two years after diagnosis) survival for common solid tumours, including breast, colorectal, head and neck, liver, lung, oesophageal, pancreatic, prostate, and stomach cancer in the UK.Using pseudonymised NHS records, we examined: 1) secular trends in the incidence of several common cancers from 2000-2019 compared to 2020-2022; and 2) short-term survival following diagnosis of the same cancers in 2000-2019 and compared to those diagnosed in 2020-2022.
Study Design
A population-based cohort study was conducted to examine the incidence and survival for nine cancers using routinely collected primary care data from the Clinical Practice Research Datalink (CPRD) GOLD database December 2022 (version 2022.12.001) in the UK between January 2000 and December 2022.
Study Participants
All patients were required to be aged 18 years or older and have at least one year of history within the database (to characterise patients on key comorbidities) and information on age and sex, excluding individuals diagnosed with the same cancer at any time in the database history.Hence, data from 1999 to 2022 were used.For incidence, the study cohort consisted of individuals present in the database from 1 January 2000 or their first day of eligibility (whichever occurred first).These individuals were followed up to whichever came first: the cancer outcome of interest, date of death, exit from the database, or 31 December 2022 (the end of the study period).For survival analysis, individuals with a newly diagnosed cancer were included.These individuals were followed up from the date of their diagnosis to either date of death, exit from the database, or end of the study period.All outcomes included both sexes, except for breast cancer (females only) and prostate cancer (males only).
Procedures
People with a diagnosis of a cancer of interest and a denominator cohort were identified from CPRD GOLD to estimate cancer incidence and overall survival.CPRD GOLD contains pseudonymised patient-level information on demographics, lifestyle data, clinical diagnoses, prescriptions, and preventive care contributed by general practitioners (GP) from the UK.It is an established primary care database broadly representative of the UK population. 12This database was mapped to the Observational Medical Outcomes Partnership common data model (CDM). 13
Outcomes
We used SNOMED CT diagnostic codes to identify incident cancer events for the nine cancers.Diagnostic codes indicative of either non-malignant cancer or metastasis were excluded, as well as diagnosis code indicative of melanoma and lymphoma occurring in the organs/sites of interest.Metastases were excluded because we intended to examine the impact of the pandemic on patients with incident cancer diagnoses that had not yet entered a clinical care pathway.Hence, the outcomes of interest were local or locally advanced cancers only.The cancer outcome definitions were reviewed with the aid of the CohortDiagnostics R package. 14This package was used to identify additional codes of interest and to remove those highlighted as irrelevant based on feedback from clinicians with oncology expertise through an iterative process.The clinical code lists used to define all cancer outcomes can be found in Supplementary 1.Additionally, a detailed description of all cancer outcomes is provided at https://dpa-pde-oxford.shinyapps.io/EHDENCancerIncPrevCohortDiagShiny.For survival analysis, mortality was defined as all-cause mortality based on date of death records.Mortality data in CPRD GOLD has been previously validated and shown to be over 98% accurate. 15
Statistical Analysis
The population characteristics of patients with a diagnosis of each cancer were summarised overall and separately for each calendar year stratum, with median and interquartile range (IQR) used for continuous variables and counts and percentages used for categorical variables.Differences in the counts of comorbidities across time were evaluated by χ 2 .Characteristics included 16 common chronic diseases, identified by clinical code lists (and reviewed by clinicians).
Annual crude incidence rates (IRs) were calculated for all cancer outcomes from 2000 to 2022.For incidence, the number of events, the observed time at risk, and the IR per 100,000 person years were summarised with 95% CIs.Annual IRs were calculated as the number of incident cancer cases as the numerator and the exact recorded number of personyears in the general population of CPRD GOLD within that year as the denominator.
Using the crude IRs, age-standardised IRs were calculated using the 2013 European Standard Population ESP2013. 16he ESP2013 serves as a standard population with a predefined age distribution where results are adjusted to match this distribution and account for differences in age structures between different populations to ensure fair comparisons.The ESP2013 provides predefined age distribution in five-year age bands; therefore, we collapsed these to obtain distributions for the 10-year age bands used in this study.We used an age distribution of 20-29 years from ESP2013 for age standardisation, as age distributions were not available for the 18-to 29-year age band used in this study.
For survival analysis, we stratified by calendar time of cancer diagnosis (2000-2004, 2005-2009, 2010-2014, 2015-2019, and 2020-2022).To estimate short-term survival at one and two years, we used the Kaplan-Meier (KM) method.Patients whose death and cancer diagnosis occurred on the same date were removed from the survival analysis (0.48%-3.64% of patients depending on cancer).To avoid re-identification, we do not report results with fewer than five cases.All results are presented in an interactive online dashboard at https://dpa-pde-oxford.shinyapps.io/CancerIncSurvCovid.
Patient and Public Involvement
No patients or members of the public were involved in the design, analysis, or interpretation of this study or the reported data, because the study aimed to examine population-level trends and patterns, rather than individual experiences or perspectives.
Ethics Approval
The protocol for this research was approved by the Independent Scientific Advisory Committee for Medicine and Healthcare Products Regulatory Agency database research (protocol 22_001843) and adhered to and complied with local relevant data protection and privacy regulations and the principles of the Declaration of Helsinki.
Results
There were 12,557,753 eligible patients aged 18 years and older with at least one year of history identified from January 2000 to December 2022 from CPRD GOLD, but 5,634,984-11,593,927 finally eligible after applying inclusion/ exclusion criteria.The attrition was largely due to not being observed in the database during the time period of interest after applying the age, sex, and history requirements.The attrition table for this study for each cancer can be found in Supplementary 2. A summary of patient characteristics of those with a diagnosis of different cancers is shown in Table 1.
As detailed in Table 1, breast cancer was the most common cancer, followed by prostate, colorectal, and lung.Males were more likely to have a cancer diagnosis apart from breast cancer and for pancreatic cancer where there were no sex differences in numbers diagnosed.Cancer diagnoses were more common with increasing age, peaking in those aged 70-79 years for all cancers, apart from those diagnosed with breast or head and neck cancer, where more diagnoses were in those aged 60-69 years.For breast, prostate, and head and neck cancers, these patients had the lowest percentages of comorbidities, whereas those with a diagnosis of liver, stomach, and lung cancers had the highest percentages of comorbidities.
Comparison of patient characteristics of those diagnosed in 2020-2022 with the other calendar-year groups (2000-2004, 2005-2009, 2010-2014, and 2015-2019) showed similar socio-demographics with very similar age and sex distributions, regardless of the year of diagnosis.Regarding comorbidities, whilst there were significant differences across all time points for various conditions across cancers, the most notable differences between 2020 to 2022 compared to previous years were greater frequencies of: 1) atrial fibrillation in colorectal, liver, lung, oesophageal, prostate, and stomach cancers; 2) cerebrovascular disease in colorectal, lung, and liver cancers; 3) venous thrombosis in colorectal, lung, oesophageal, and prostate cancers; 4) COPD in colorectal, head and neck, and liver cancers; 5) type 2 diabetes in colorectal, head and neck, liver, lung, pancreatic, prostate, and stomach cancers; 6) hypertensive disorder in head and neck and liver cancers; 7) chronic liver disease in liver and prostate cancers; 8) osteoarthritis in liver, lung, pancreatic, and prostate cancers; 9) depressive disorder in liver and prostate cancers; 10) pulmonary embolism in lung and oesophageal cancers; and 11) gastrointestinal haemorrhage in oesophageal cancers.Detailed patient characteristics stratified by calendar year of diagnosis in five-year strata can be found in Supplementarys 3.1-3.9.
Regarding secular trends in the incidence of cancer, annual age-standardised IRs increased from 2000 to 2019 for all cancers except breast, colorectal, oesophageal, and stomach cancer (Figure 1).Apart from pancreatic cancer, IRs decreased for all cancers in 2020 and recovered to different extents in 2021-2022.For breast, colorectal, oesophageal, and pancreatic cancers, IRs in 2021 were higher than 2019, whereas for head and neck, liver, lung, prostate, and stomach cancers, IRs were still lower in 2021 than before the pandemic in 2019.Results showing the crude annualised IRs can be found in Supplementary 4.
The KM survival curves for all nine cancers from the date of diagnosis to two-year follow-up are shown in Figure 2. Individual plots for each cancer can be found in Supplementarys 5.1-5.9.The numbers at risk, events, and censoring at each of the time points in the KM plots for each cancer shown in Figure 2 are included in Supplementary 6. Cancer survival curves depicted in Figure 2 are consistent with the one-and two-year survival probabilities shown in Table 2. Survival trends tended to improve in the years 2000-2019, but then declined for breast, head and neck, oesophageal, and pancreatic cancers after their diagnosis in 2020-2022.
Statement of Principal Findings
The incidence of cancer diagnoses in the UK decreased in the first year of the pandemic (2020), recovering to different extents in 2021 for most cancers.This was more obvious for breast, colorectal, lung, and oesophageal cancer, with rates of new diagnosis in 2021 and 2022 higher than those recorded immediately before the pandemic in 2019.One-and two-year survival after a diagnosis of cancer increased in the years 2000-2019 for most cancers and more clearly for cancers that benefit from novel treatments including antivirals (eg, liver cancer) and immunotherapies (eg, lung cancer).Cancers included in national screening programmes (eg, breast, colorectal) also improved prognosis in these same years, likely due to earlier diagnosis.
In contrast with observable improvements over the previous 20 years, the first two years of the pandemic led to a decline, albeit minimal, in short-term survival for some cancers (breast, head and neck, colorectal, oesophageal, and pancreatic cancer) in the UK.This decline was most pronounced for colorectal cancers, with reductions in survivorship equivalent to the return to mortality rates seen in the first decade of the 2000s.Although only a difference of 1.4%, this represents a substantial number of patients for whom survival could have been improved through early detection and treatment, which were halted during the pandemic. 3,17We found no evidence that this decrease in survivorship could be due to differences in socio-demographics: patients diagnosed with cancer in 2020-2022 were of similar age and sex and had similar comorbidities compared to those diagnosed in previous years.However, it should be noted that there was a greater frequency of some comorbid conditions in patients diagnosed during the pandemic compared to previous years, and this may account for decreases in survival.In line with this, there may be competing causes of death other than cancer, particularly due to COVID-19 infection during this time.
Whilst it is pertinent to account for the impact of COVID-19 infection on mortality in the cancer population, cause of death is not captured by the CPRD dataset.As such, it was not possible to quantify this potential competing cause of death from COVID-19.Future research should account for this so that it is possible to delineate the impact of the pandemic on cancer survival whilst ruling out COVID-19 infection itself as a contributing cause.
Strengths of the Study
The large sample and over 20 years of follow-up provided by CPRD GOLD make this a unique dataset for longitudinal analyses.Cancer incidence before the pandemic reported here is in line with national data from Cancer Research UK statistics for all cancers, providing confidence in the validity of our estimates. 18The high validity and completeness of mortality data, with over 98% accuracy when compared to national mortality records, 15 allowed us to demonstrate the impact of the COVID-19 pandemic on short-term survival, one of the key outcomes in cancer care.Weaknesses of the Study Our study also had limitations.First, we used primary care data without linkage to cancer registry, potentially leading to misclassification and delayed recording.However, previous validation studies have shown high accuracy and completeness of cancer diagnoses in primary care records. 19Second, our use of primary care records precluded us from studying tumour histology, staging, or cancer therapies, which can all impact survival.Third, the composition of the CPRD GOLD database has changed over time.With the advent of the CPRD AURUM database, some English practices were transferred out of GOLD into AURUM from 2021.Therefore, the data from 2021 onwards are more representative of Scotland and Wales and less representative of England and Northern Ireland than earlier datacuts.
Research in Context
In line with our results, UK cancer statistics demonstrate the increasing trend in cancer incidence over recent decades. 18,20easons for these increases are multifaceted and vary per cancer type, but generally include diagnostic testing (eg, PSA for prostate cancer), public awareness of symptomology (eg, for breast cancer), and an ageing population. 20A notable trend observed is a significant increase in breast cancer incidence during 2004-2005.One potential explanation for this could be the implementation of the Quality and Outcomes Framework in 2004.This evaluates the performance of general practices across various disease areas, including cancer, and offers financial incentives for meeting specific quality targets.Consequently, there may have been increased focus on screening, diagnostics, and reporting of cancer cases during this period.Moreover, from 2001 to 2004, many screening units in the UK expanded their screening programs to include women aged 65-70 years. 21This expansion could have contributed to the observed surge in cancer diagnoses during this time frame.Unsurprisingly, the COVID-19 pandemic halted screening programmes and diagnostic appointments, and may have altered patients' health-seeking behaviour in an attempt to contain the virus. 3These alterations led to reduced cancer diagnoses, as evidenced by our results (with the exception of pancreatic cancer), as well as other European data. 11,22,23owever, there may be other reasons for the reduction in cancer diagnoses during the pandemic.For example, reduced exposure to certain cancer risk factors due to relatively healthier lifestyles during lockdown (such as decreased smoking, decreased alcohol consumption, and healthier dietary and sleep patterns 24,25 ) may have halted tumour development.An alternative explanation is that undiagnosed cancer patients may have died from COVID-19 infection.Finally, delays or inaccuracies in cancer data collection and reporting may affect the apparent incidence.Regardless of the explanation, our results contrast with data from the United States showing an increase in diagnoses of lung, colorectal, pancreatic, breast, and prostate cancers in 2020 compared to 2019, 26 and illustrate the selective channelling of healthcare resources in the UK during the first 2 years of the pandemic.Not surprisingly, 2021-2022 saw a relative increase in incidence of these cancers compared to 2020, which suggests attempts to compensate for those diagnoses missed during the pandemic.Our data for the first time show that cancer incidence have largely recovered since 2020, yet rates of diagnosis need to continue to exceed expected rates in the forthcoming years to account for the backlog in diagnoses accumulated in 2020.
Possible reasons that the incidence of pancreatic cancer did not decrease during the pandemic compared to other cancers include the fact that pancreatic cancer is usually diagnosed at an advanced stage, as there are not good predictors for screening and early diagnosis.Cancer arising at the pancreatic head is usually diagnosed when jaundice appears due to compression of the bile duct.Patients with such symptoms as jaundice attended hospital during the pandemic, and the diagnosis was generally determined without a relevant delay.Cancers arising at the body or tail of the pancreas present non-specific symptoms and are usually diagnosed at a very advanced stage, when the patient has very severe symptoms of weight loss and debility.In such conditions, CT scans for diagnosis during the pandemic were probably prioritised and diagnoses not remarkably delayed.In fact, it is possible that they were even scheduled sooner than usual, because CT scans for other non-urgent indications were delayed and there was greater availability of the scanner.
One major implication of reductions in cancer incidence is that diagnoses and treatments have been delayed, affecting patient survival.This may be particularly prominent for cancers included in screening programmes, such as colorectal cancers.Whilst the literature on survival estimates post-pandemic is sparse, some global estimates align with our UK-based estimates demonstrating the impact of diagnostic delays and reduction in care/availability of treatments on cancer survival, with an increase in total deaths from most cancers during 2019-2021. 26That said, during 2020-2022 the one-year survival rate of liver, lung, breast, and prostate cancers, as well as the two-year survival rate of colorectal, lung, liver, pancreatic, prostate, and stomach cancers, did not decrease.There are many possible explanations as to why this may be.For example, this may be because these particular cancers benefited from increased remote monitoring or adaptation of treatment protocols during and immediately following the pandemic.Alternatively, patients may have been more motivated during the COVID-19 pandemic to adhere more strictly to treatment plans, adopt healthier lifestyles, and prioritize self-care measures to optimize their health and recovery.
Conclusion
Taken together with previous research, our results show evidence that short-term survival from cancer was impacted by the management of the COVID-19 pandemic in the UK.The combination of a decline in diagnoses followed by an increase in short-term mortality for many cancers and more clearly for colorectal malignancies provide evidence of delays in diagnosis during the first two years of the pandemic.However, other factors, such as reduced care/unavailability of certain cancer treatments, and COVID-19 infection, may have contributed to reduced survival of those diagnosed with cancer during the pandemic.Our findings illustrate the need for an immediate and intense investment from the NHS to resolve the current backlog in cancer screening and diagnostic procedures to improve cancer survival.Datasets with longer follow-up are now needed to fully comprehend the impact of COVID-19 on long-term cancer care and survival.
Figure 1
Figure 1 Age-standardised annual incidence (per 100,000 person years) for nine cancers from 2000 to 2022 (red line indicating start of COVID pandemic in 2020).Grey bands indicate 95% CIs.
Figure 2
Figure 2 Kaplan-Meier survival curves for all nine cancers stratified by calendar time of cancer diagnosis.Shaded bands indicate 95% CIs.
Table 1
Baseline characteristics of patients at the time of cancer diagnosis for this study from 2000-2022 | 2024-06-13T15:02:58.078Z | 2024-06-01T00:00:00.000 | {
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227101896 | pes2o/s2orc | v3-fos-license | Evaluation of a Conservative Pharmacist-led U-500R Insulin Management Protocol in the Primary Care Setting
Objective The objective of this quality assurance study is to evaluate the impact of a conservative, pharmacist-led, U-500R insulin management protocol on diabetes control (A1c) and total daily dosage requirements between August 2016 and August 2018. Methods This was a retrospective chart review of adult patients, aged 18 to 79, with type 2 diabetes and managed with insulin, at 2 federally qualified healthcare clinics in Denver, Colorado. To determine if our conservative pharmacist-led U-500R insulin management protocol impacted efficacy and total daily dosage requirements when converting patients from U-100 to U-500R insulin, we compared the most effective dose of U-500R (defined as the total daily dose (TDD) of U-500R insulin at A1c goal or the lowest tolerated A1c) to the baseline A1c and TDD of U-100 insulin at time of conversion. Results Following conversion of U-100 to U-500R insulin, patients required an average of 21 fewer units of insulin with U-500R than U-100 and achieved an average A1c of 7.2% which reflected a reduction of 3.5 points from baseline. Five patients (62.5%) achieved A1c goal per ADA guidelines, and all patients achieved at least a 1.7 point reduction in A1c, with 1 patient achieving a 6.7 point reduction. Two patients (25%) were still in the process of U-500R titration at the time of data collection, and 1 patient (12.5%) did not achieve goal A1c while under pharmacy management at these clinics. Four of the five patients who achieved A1c goal did so with an overall reduction in total daily insulin dose (average of 57.5 units less than original U-100 dose) resulting in an average A1c decrease of 3.6 points.
Introduction
Achieving optimal blood glucose (BG) control in patients with Type 2 diabetes mellitus and severe insulin resistance can be challenging. These patients often require large volumes of U-100 insulin which may lead to altered absorption and leakage during administration. 1 An alternative approach to large insulin doses is to transition from U-100 insulin to human (regular) 500 units/mL insulin (U-500R). 2 This option is often desirable since smaller volumes are required of U-500R, as the insulin is 5 times more concentrated than U-100 insulin. Because smaller volumes are injected with the U-500R product, insulin leakage can be minimized, thus improving insulin absorption, patient comfort, and A1c. Most patients will have improved glycemic control using twice daily U-500R, but the precise dosing regimen depends on the total daily insulin requirements of the patient. 3,4 However, because U-500R is highly concentrated, many providers in primary care settings will not prescribe U-500R, but prefer to refer patients to be managed by endocrinology. When patients are unable to receive specialty care from endocrinology due to financial constraints or when endocrinology practice sites are closed to new patients, management of diabetes remains in the primary care setting. This situation occurred at 2 federally qualified healthcare centers where patients who had severely insulin resistant, uncontrolled diabetes were managed by their primary care provider, because they were unable to access endocrinology specialty care. Fortunately, these 2 clinics provided interprofessional patient care, which included a clinical pharmacist who split her time between the 2 practice sites. In 2013, a decision was made to develop a pharmacist-led program to help primary care providers address this gap. A U-500R insulin protocol for clinic use was created to guide insulin conversion, titration and management, since manufacturer dosage guidance was not available at that time other than the package insert that stated U-500R is to be dosed 2 to 3 times daily. In 2013, the manufacturer provided no guidance for converting patients from U-100 insulins to U-500R.
The clinical pharmacist collaborated with the clinic's primary care providers and diabetes educators to develop a Collaborative Drug Therapy Management (CDTM) pharmacist-led U-500R insulin management program. This program included a conservative dosing conversion process as part of the U-500R insulin management protocol which prioritized safety, because U-500R is the most concentrated insulin available on the US market. The U-500R insulin management program was implemented in 2013 at both clinics and has been reserved for patients who have severe insulin resistance and are unable to schedule an appointment with endocrinology (either due to endocrinology clinics not accepting additional Medicaid patients or based on financial constraints for patients who are uninsured). Criteria for initiation into the U-500R insulin management program includes patients who have minimal or no mental cognitive impairment, are receiving 200 units or more of insulin daily, can recognize and appropriately manage hypoglycemic episodes and are adherent with clinic visits. Per protocol, upon initiation of a patient into the U-500R insulin program, a pharmacist calculates the patient's total daily U-100 insulin dose and reduces that dose by 20% to determine the starting dose of U-500R. Patients are instructed to inject 60% of the total daily U-500R dose 30 minutes before breakfast and the remaining 40% 30 minutes before dinner. Patients are required to demonstrate appropriate insulin administration technique prior to initiating U-500R, and the pharmacist uses the teach-back method to ensure patient understanding. Patients are then followed by pharmacy services for insulin titration based on blood glucose values and tolerability.
More recently, in 2015, the manufacturer of U-500R has provided a dosing conversion algorithm, based on a study by Hood et al., the U-500 Initiation Trial. 2,3 The algorithm recommends initiating U-500R at 100% of the patient's U-100 total daily dose (TDD) if their A1c is greater than 8% and the mean self-monitored plasma glucose ≥183 mg/dL during the week preceding the initiation of U-500R. Despite the current availability of a dosing conversion algorithm provided by the manufacturer, our clinics have continued to use the conservative pharmacist-led U-500R dosing protocol which empirically reduces the total daily insulin dose by 20% regardless of A1c values. This decision was made to ensure patient safety, as there is a paucity of data supporting the 1:1 dosing conversion for patients with an A1c greater than 8% as recommended by the manufacturer. Thus, the objective of this quality assurance study is to evaluate the impact of a conservative pharmacist-led U-500R insulin management protocol on diabetes control (A1c) and total daily dosage requirements.
Methods
This was a retrospective chart review of adult patients managed by clinical pharmacy services at 2 underserved primary care clinics in Denver, Colorado. This initiative was approved as exempt by the Colorado Multiple Institutional Review Board. Patients were included if they were aged 18-79 and were using U-500R to manage type 2 diabetes between August 2016 and August 2018. Patients were excluded if they were initiated on U-500R prior to receiving care at one of these 2 clinics, if the patients' U-500R insulin regimen was not managed by pharmacy services or if U-500R was newly initiated and had not yet been titrated. Patient charts were reviewed by a designated clinical pharmacist at both study sites. Data collection included patient demographic information (ie, age, sex, weight, BMI), A1c goal based on the ADA guidelines, TDD of U-100 insulin at time of conversion, initial dose of U-500R, and most effective dose of U-500R (defined as the TDD of U-500R insulin at the time of achieving A1c goal or the lowest A1c).
To determine if our conservative pharmacist-led U-500R insulin management protocol impacted efficacy and total daily dosage requirements when converting patients from U-100 to U-500R insulin, we compared the most effective dose of U-500R to the baseline A1c and TDD of U-100 insulin at time of conversion. Descriptive analysis was completed for all variables evaluated except for data regarding A1c control which was analyzed using Fisher's exact test with an a priori significance level of 0.05.
Results
Thirteen patients met initial inclusion criteria. Four patients were excluded because their U-500R dosing was not managed by pharmacy services and another patient was excluded because U-500R had been initiated but not yet titrated at the time of chart review-leaving a total of 8 patients included in the case series. The average age of patients was 59 years (50% male), and the majority of patients had a goal A1c of less than 7%. At baseline, patients had an average A1c of 10.7% and required an average of 258 units of U-100 insulin (Table 1), with no patients having an A1c below 8% at the time of conversion. The U-500R doses were titrated according to our clinic protocol until goal A1c was achieved or further titration could not be tolerated. According to clinic protocol, all patients were initiated at 80% of their total daily U-100 pre-conversion insulin dose. Five patients (62.5%) achieved maximum A1c reductions on a U-500R dose that was lower than their pre-conversion U-100 insulin TDD (Figure 1). Of those patients, the average reduction in TDD was 18% at the time of maximum titration with 4 of the 5 achieving goal A1c. In the 3 patients who required more units of U-500R compared to the pre-conversion U-100 dose, the average increase was 30 units, or a 12% increase from pre-conversion U-100 TDD (Figure 2). There was a non-significant reduction in A1c between patients who required a lower dose of U-500R versus, those who required a higher dose (−3.2 vs −3.9 P < .8088). Overall, patients required an average of 21 fewer units of U-500R than of U-100 and achieved an average A1c of 7.2% which reflected a reduction of 3.5 points from baseline ( Table 2). Five patients (62.5%) achieved A1c goal per ADA guidelines, and all patients achieved at least a 1.7 point reduction in A1c, with 1 patient achieving a 6.7 point reduction. Two patients (25%) were still in the process of U-500R titration at the time of data collection, and 1 patient (12.5%) did not achieve goal A1c while under pharmacy management at these clinics. Four of the five patients who achieved A1c goal did so with an overall reduction in total daily insulin dose, an average of 57.5 units less than original U-100 dose resulting in an average A1c decrease of 3.6 points.
Discussion
The results of this study illustrate how a conservative, pharmacist-led, U-500R insulin management program was effective in improving A1c in the majority of patients and at lower total daily dosage requirements of U-500R compared to U-100 insulin. On average, A1c was reduced from 10.7% to 7.2% following the conversion of U-100 to U-500R. Most importantly, 62.5% of patients achieved maximum A1c reductions on a final U-500R dose that was lower than their pre-conversion U-100 insulin TDD. Specifically, 80% of the patients who achieved their A1c goal did so with a reduction in total daily insulin dose by an average of 57.5 units less than their original U-100 dose. Although not all patients reached their A1c goal, patients who had an overall improvement in A1c required a lower total daily dose of insulin by 21 units. All patients included in the study were highly insulin resistant with uncontrolled type 2 diabetes and were unable to access specialty care with endocrinology. Had the providers decided to initiate U-500R based on the current manufacturer's algorithm, all of the patients included in this study would have qualified for a 1:1 conversion from U-100 to U-500R, and it is possible that harm from hypoglycemic events may have occurred in these patients. Having a conservative, pharmacist-led, U-500R insulin management program allowed for patient access to a highly concentrated insulin as well as safe and effective management of each of these patients.
There is a paucity of literature demonstrating the role of clinical pharmacists in the management of U-500R insulin other than providing collaborative care (eg, patient education, clinical consults) which primarily occurs in the hospital setting. This study is unique, as it illustrates the outcomes of a pharmacist-led U-500R insulin management program on reductions in patient A1c and on U-500R insulin dosage requirements in the primary care setting, where pharmacists can independently manage U-500R under protocol. However, it is important to note that the role of the clinical pharmacist extends beyond the insulin conversion and management of patients transitioned to U-500R. Similar to the pharmacist-led U-100 rapid insulin titration program at each of these clinics, patients enrolled in the pharmacist-led U-500R insulin management program receive intensive lifestyle coaching on a weekly or bi-weekly basis. These coaching sessions likely enhance positive patient outcomes as related to diabetes control as well as safety. Because U-500R is a highly concentrated form of insulin, close follow-up upon conversion of U-500R is critical. With more frequent monitoring, patients also receive more intense lifestyle monitoring, education, coaching and reinforcement. Improved lifestyle choices may lead to weight loss, which in turn often leads to reduced insulin requirements. Reduced insulin requirements in patients who are making positive lifestyle changes is another reason that a conservative dosing conversion algorithm may be important.
In the development of the pharmacist-led U-500R insulin management program, emphasis was placed on ensuring the formation of a conservative protocol, as there is a lack of published research evaluating dosing algorithms used to convert patients from traditional U-100 insulin regimens to U-500R insulin. It was assumed that patients may need a lower amount of U-500R compared to U-100 insulin which is why we compared the U-100 and U-500R total daily literature to support the outcomes of these dosing algorithms, safety was determined to be uncertain. Other clinical reviews and studies proposed methods to transition patients to U-500R, but these suggestions were based on small case reports or case series. [5][6][7] Published studies evaluated efficacy and side effects of U-500R insulin, but to our knowledge, there are no published studies whose purpose was to evaluate conservative dosage conversion strategies.
Our study demonstrates that even with a 20% reduction in TDD of insulin during the transition from U-100 to U-500R, some patients required a further dose reduction. Without the initial dose reduction, it is possible that our patients may have suffered consequences from excessive hypoglycemia. We hope the results from this case series will help guide other primary care clinicians who are considering the use of U-500R for patients who are unable to access specialty care until more literature is available to evaluate the use of U-500R insulin in the primary care setting.
Limitations
A limitation of this study is the sample size, with only 8 patients who qualified for analysis. Additionally, the data was retrospective and descriptive. Furthermore, hypoglycemic episodes were not analyzed, so safety concerns are speculative. Lastly, although coaching sessions are provided to patients managed by clinical pharmacy services, we did not assess if patients were managed by pharmacy prior to the transition to U-500R. As such, coaching sessions may have additionally contributed to the reduction in A1c and insulin requirements for some patients studied.
We realize the results of this study may not be applicable for most primary care clinics but are optimistic that this data could provide some guidance for other clinics that manage underserved patients who may not have access to specialty care.
Conclusion
The conservative pharmacist-led U-500R dosing protocol was an effective tool to transition patients from U-100 to U-500R insulin in the primacy care setting, as 62.5% of the patients converted to U-500R per protocol had a final U-500R dose lower than their pre-conversion U-100 TDD while achieving an average A1c reduction of 3.5 points. Furthermore, 66% of patients who achieved their goal A1c did so using fewer units of U-500R than of U-100.
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. | 2020-11-22T14:09:29.747Z | 2020-11-01T00:00:00.000 | {
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40259275 | pes2o/s2orc | v3-fos-license | Selective use of laparoscopy in nonpalpable undescended testes
46 patients diagnosed with nonpalpable UDT between 2007 and 2012 who underwent an inguino-scrotal ultrasound preoperatively. We analyzed correlations between radiological and surgical findings. Results: A total of 46 patients (53 UDT), median age 14 months (quartile 1 st : 7; 3 rd : 80) were included. Ultrasound localized the testis as intracanalicular in 24/53 (45.2%), intraabdominal in 10/53 (18.8%), scrotal in 1/53 (1.8%), and could not localize 18/53 (33.9%) testes. In 35/53 (66%) testes, the ultrasound location correlated with the surgical findings ( P < 0.001). Ultrasound detection showed 96% sensitivity and 56% specificity for intracanalicular testes. Conclusion: The use of preoperative ultrasound in this series was helpful in identifying the location of nonpalpable testes in children. In particular, the ultrasound finding of an intracanalicular testis may preclude the need for laparoscopy.
INTRODUCTION
Isolated cryptorchidism is one of the most common congenital anomalies, affecting approximately 3% of full-term male infants and 30% of premature infants. [1,2] Between 3 and 6 months of age, the incidence of undescended testis (UDT) decreases to 1-1.5% due to spontaneous descent. The most useful determinant of management for cryptorchidism is whether the testis is palpable on physical examination. It can, however, be difficult to accurately determine the exact location of the testis by palpation; body habitus, testicular position, and compliance of the child may impede the physical examination. Approximately 20% of UDT are nonpalpable, and these testes may be intra-abdominal, inguinal, ectopic or absent. Most intra-abdominal testes are found within a few centimeters of the internal ring, however approximately 20-40% of nonpalpable testes are absent upon surgical exploration. [3] The surgical management for nonpalpable testes is diagnostic laparoscopy. [4] The evidence shows that radiologic studies to localize the testis are of very little value, with sensitivity and specificity of approximately 45% and 78%, respectively. [5][6][7] In one series, it was reported that ultrasonography had no value in the diagnosis of the nonpalpable testis. [8] The objective of this study was to determine if ultrasonography is accurate as a preoperative tool to localize nonpalpable testes in order to prevent unnecessary laparoscopy.
METHODS
We retrospectively collected demographic, radiological, and surgical data for patients diagnosed with nonpalpable UDT, who underwent an inguino-scrotal ultrasound preoperatively between June 2007 and June 2012 at our institution. We examined correlations between radiological and surgical findings for these patients.
DISCUSSION
The aim of surgical management of the UDT in young boys is to preserve spermatogenesis [9] and allow for screening and detection of any malignant transformation by performing an early orchiopexy. [10] Approximately 80% of UDT are palpable, and they are managed by an orchiopexy performed through a small inguinal incision. Nonpalpable testes, on the other hand, represent a diagnostic and therapeutic challenge. Such testes may be in an intra-abdominal location, intracanalicular, in an ectopic site or vanished due a prenatal torsion. Depending on the testicular viability and length of the spermatic cord, during exploration, the testicle is either secured in the scrotum or removed. The current initial surgical procedure of choice for the nonpalpable UDT is a diagnostic laparoscopy. [11] The findings on laparoscopy dictate the next step of management; either a one-or two-stage laparoscopic assisted orchiopexy, a laparoscopic orchiectomy or an inguinal exploration if the vas deferens and spermatic vessels pass through the internal ring into the inguinal canal.
The laparoscopic procedure could be avoided in some cases if a preoperative imaging study could reliably localize a nonpalpable testis, which is situated in the inguinal canal. This would save time and resources as well as minimizing the operative risk to patients undergoing these procedures. Laparoscopy is widely and safely used by urologists, however, it carries some risks, especially in children given their size and the limited workspace in the peritoneum, as well as additional costs for the health care system. Vascular, bowel, and solid organ injuries are among the possible complications seen with laparoscopy. [12][13][14] We propose that modern day high-resolution ultrasound, performed by experienced sonographers, can be used as a tool to localize the nonpalpable inguinal UDT.
In this series, all 46 boys with UDT were examined by a pediatric urologist, and underwent ultrasonography at a tertiary pediatric hospital; all included patients underwent examination under anesthesia together with a diagnostic laparoscopy. We correlated the ultrasonographic finding in these boys with the surgical findings and found that in 66% of testes, ultrasound localization correlated with surgical findings (P < 0.001). The overall sensitivity and specificity of diagnostic ultrasonography were 96% and 56%, respectively, for testes in the inguinal canal. Thus, we feel that ultrasound localization of a nonpalpable inguinal UDT is a useful adjunct to the surgeon's armamentarium. Our findings suggest that visualization of a testis in the inguinal canal by ultrasound can allow the surgeon to proceed with confidence knowing that an inguinal exploration will be all that is required, and laparoscopy is not necessary.
We acknowledge that in most of the published literature, ultrasound performs poorly in localizing the nonpalpable UDT. [15] However, such studies were focused on using the ultrasound study to eliminate the surgical procedure, with a 35% probability of missing an intra-abdominal testis, which we are not proposing in this study. Our focus was using ultrasound for the detection of the nonpalpable intracanalicular testis, thus giving more confidence to the surgeon to proceed to inguinal exploration. When the testis is nonpalpable and seen by ultrasound as intra-abdominal or not visualized, diagnostic laparoscopy should be undertaken. [16] Two recently published studies support our approach to the nonpalpable UDT. One study examined the use of ultrasound in 49 boys with 60 UDTs and revealed that ultrasound identified 97% of UDTs located in the inguinal canal, with 77% of these being palpable. Ultrasound was not found to be useful in identifying intra-abdominal testes. [17] A second prospective study used high-resolution ultrasound to examine 40 boys with 52 UDTs, comparing the findings to those at surgical exploration. Up to 91% of palpable and 87% of nonpalpable testes were identified, with an overall sensitivity of 90%, specificity 33%, positive predictive value 96% and negative predictive value 17%. [18] The number of subjects, as well as the retrospective design, limits our study. Due to the retrospective data collection there is a possibility of bias such as classification and selection of nonpalpable testes. A prospective study with a larger subject numbers will be required to confirm these findings.
CONCLUSION
This series supports the use of diagnostic ultrasonography for preoperative assessment of patients with nonpalpable UDT. For those in whom an intracanalicular testis is seen on US, one may avoid laparoscopy and proceed with inguinal exploration. For patients in whom the testis is visualized intra-abdominally or not at all, laparoscopy should be undertaken. | 2018-04-03T05:05:21.171Z | 2016-01-01T00:00:00.000 | {
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18465633 | pes2o/s2orc | v3-fos-license | Chinese
Functional magnetic resonance imaging was used to explore the neural correlates of semantic judgments in a group of 8- to 15-year-old Chinese children. Participants were asked to indicate if pairs of Chinese characters presented visually were related in meaning. The related pairs were arranged in a continuous variable according to association strength. Pairs of characters with weaker semantic association elicited greater activation in the mid ventral region (BA 45) of left inferior frontal gyrus, suggesting increased demands on the process of selecting appropriate semantic features. By contrast, characters with stronger semantic association elicited greater activation in left inferior parietal lobule (BA 39), suggesting stronger integration of highly related features. In addition, there was a developmental increase, similar to previously reported findings in English, in left posterior middle temporal gyrus (BA 21), suggesting that older children have more elaborated semantic representations. There were additional age-related increases in the posterior region of left inferior parietal lobule and in the ventral regions of left inferior frontal gyrus, suggesting that reading acquisition relies more on the mapping from orthography to semantics in Chinese children as compared to previously reported findings in English.
features (Fletcher et al., 2000). In support of this, many studies show greater inferior frontal gyrus activation in more diffi cult semantic tasks and in tasks with increased retrieval or selection demands. These studies include high vs. low requirement for selection among alternatives (Thompson-Schill et al., 1997, weak vs. strong association strength (Wagner et al., 2001;Chou et al., 2006a,b), naming low vs. high familiarity objects (Whatmough et al., 2002), generating novel vs. repeated base nouns (Seger et al., 2000), naming low vs. high agreement pictures (Kan and Thompson-Schill, 2004), deep vs. shallow processing of words (Fujii et al., 2002) and producing words for pre-specifi ed semantic categories vs. over-learned letter sequences (Gurd et al., 2002).
A review article recently proposed different cognitive functions for sub-regions of the inferior frontal gyrus (Badre and Wagner, 2007). The anterior ventral region of left inferior frontal gyrus (BA47) may support controlled retrieval of stored semantic representations, whereas the mid-ventral region of left inferior frontal gyrus (BA 45) may support increased selection demands to process active representations. Previous studies using association strength have found greater activation for weaker association pairs in both the anterior ventral region and the mid-ventral region of left inferior frontal gyrus (BA 45,47) (Badre et al., 2005;Chou et al., 2006a,b). For weaker association pairs, participants may need controlled access to stored conceptual representations to seek for existing associations in verbal semantic memory. Moreover, participants may require a selection process
INTRODUCTION
Language is acquired without much effort and is associated with maturational changes in the brain (Friederici, 2006). The nature of developmental changes in human cerebral functional organization for language is a core issue in cognitive neuroscience (Brown et al., 2005). This paper focuses on the developmental changes in the neural substrate for semantic processing in reading, which may be somewhat different than spoken language. Emerging evidence of the neural correlates of semantic development mainly comes from English and other alphabetic languages. This paper uses a semantic association task to explore the age effects on the neural correlates of semantic processing in a logographic language, Chinese. The semantic association task has been used to understand the functional architecture of word recognition, identifying brain regions for semantic processing in English in left inferior frontal gyrus (BA 45,47), left posterior middle temporal gyrus (BA 21), and left inferior parietal lobule (BA 39,40) in adults (Fletcher et al., 2000;Booth et al., 2002) and in children (Chou et al., 2006a,b).
The role of inferior frontal gyrus in semantic processing has been explored in English by comparing activation to semantic association judgments involving strongly related pairs (e.g., king-queen) versus weakly related pairs (e.g., net-ship). Weaker association produced greater activation in left inferior frontal gyrus as compared to stronger association. Greater activation in left inferior frontal gyrus was suggested to result from the diffi culty of selecting appropriate semantic features, as weakly related pairs share few semantic that operates post-retrieval to resolve competition among active representations during association judgments.
Studies in English have also shown that weaker association strength between words results in greater activation in left middle temporal gyrus (Chou et al., 2006a;Wible et al., 2006). This region has been implicated in the representation of verbal semantic information (Booth et al., 2002;Blumenfeld et al., 2006). Greater activation in this region for weaker association pairs may result from a more extensive access to semantic representations in order to identify distant relationships . In contrast, studies in English have shown that word pairs with stronger association result in greater activation in left inferior parietal lobule (Chou et al., 2006a,b;Raposo et al., 2006). Some studies have interpreted the left inferior parietal lobule as involved in feature integration and semantic categorization to form a coherent concept so that relationships between words can be determined (Smith, 1995;Grossman et al., 2003). Left inferior parietal cortex has also been suggested to support the integration of lexical input into the larger units during semantic processing (Lau et al., 2008). Stronger association pairs may involve greater integration because there are more overlapping features between the words or because the shared features are more characteristic of each of the words (Chou et al., 2006a,b). Thus, if there are several overlapping features between words, then semantic integration processes are effectively engaged in the inferior parietal lobule and this results in greater activation in this region. However, if there are few or no overlapping features, then the inferior parietal lobule will not be engaged because it is less or not possible to integrate these features.
Chinese is different from English in the nature of the mapping between orthography and semantics as well as the mapping between orthography and phonology. English has an arbitrary relationship between orthography and semantics at a mono-morphemic level, whereas many Chinese characters are derived from pictures representing meanings or encode meanings by including a semantic radical. There are approximately 200 semantic radicals in Chinese and these units of characters give a clue to the meaning of the character (e.g., category). Thus, Chinese has a more direct mapping between orthography and semantics than English. In support of this, evidence from event-related potential measures suggests earlier involvement of semantics in Chinese compared to English (Zhang et al., 2006). Moreover, spoken Chinese is highly homophonic, with a single syllable shared by many characters. When learning to read, a Chinese child is confronted with the fact that a large number of written characters correspond to the same syllable, and phonological information is insuffi cient to access semantics of a printed character. There are phonetic radicals, but most of them (about 60%) provide inconsistent information regarding pronunciation. It is important to explore whether the unique linguistic features of Chinese infl uence developmental changes in the neural substrate for semantic processing. Based on the more systematic relationship between orthography and semantics and less systematic relationship between orthography and phonology as compared to English, one may expect a larger role for semantics in processing Chinese characters.
Previous English studies examining developmental differences at word level during semantic processing tasks have shown age-related increases in the dorsal region of left inferior frontal gyrus (Schapiro et al., 2004;Brown et al., 2005;Schmithorst et al., 2006;Szafl arski et al., 2006), the anterior region of left inferior parietal lobule (Chou et al., 2006a), and the posterior region of left middle temporal gyrys (Chou et al., 2006a,b;Szafl arski et al., 2006). Limited neuroimaging studies have examined developmental differences in the neural correlates of Chinese language processing (Cao et al., 2009, in press), showing increasing reliance on brain areas involved in visuoorthographic processing and concomitant decreases in reliance on phonology in spelling and rhyming tasks in the visual modality. Reading for meaning places greater demands on the mapping from orthography to semantics at word level in Chinese (Leck et al., 1995;Zhou and Marslen-Wilson, 2000;Feng et al., 2001), and therefore one may expect that the unique structure of Chinese infl uences developmental trajectories. Reading development of Chinese may be characterized by decreased reliance on phonology due to many homophones in Chinese, making it more effi cient to access meaning through the connection between orthography and semantics rather than the connection of orthography to phonology to semantics (Peng et al., 1985;Song et al., 1995;Meng et al., 2007).
The current study examined the neural substrate of developmental changes during semantic processing in Chinese children (8-to 15-year olds). We manipulated semantic relatedness by varying the 'free association strength' between pairs of visually presented Chinese characters (Hue et al., 2005). From aforementioned studies, we expected weaker association to produce greater activation in the ventral region of left inferior frontal gyrus, and stronger association to produce greater activation in left inferior parietal lobule. Moreover, we investigated whether there were similarities and differences on reading for meaning in the developing brain in Chinese as compared to those reported in English, with an attempt to differentiate between brain processes inherent across languages from those specifi c to the unique structure of a language.
PARTICIPANTS
Thirty-three native speakers of Chinese (mean age = 12.3, standard deviation = 1.8, 16 females) in Taiwan participated in the functional magnetic resonance imaging (fMRI) study. The participants were all monolingual. The distribution of ages and genders is shown in Table 1. Parents were given an informal interview to insure that their children met the following inclusionary criteria: (1) right-handedness, (2) normal hearing and normal or corrected-to-normal vision after an examination of vision by experimenters, (3) free of neurological disease or psychiatric disorders, (4) not taking medication affecting the central nervous system, (5) no history of intelligence, reading, or oral-language defi cits, and (6) no learning disability or attention defi cit hyperactivity disorder. After the administration of the interview, informed consent was obtained. Informed consent procedures were approved by the Institutional Review Board at the National Taiwan University Hospital. Standardized intelligence testing was then administered, using the Wechsler Intelligence Scale for Children the stimuli perceptually. For the perceptual control condition, trials consisted of a solid square (500 ms), followed by the fi rst noncharacter (800 ms), a 200-ms blank interval, and the second noncharacter for 3000 ms. Participants determined whether the pair of stimuli were identical or not by pressing a yes or no button with their right hand. There were also 24 baseline events as 'null' trials so that we could better deconvolve the response to the lexical and perceptual trials. The participant was instructed to press a button when a solid square (1300 ms) at the center of the visual fi eld turned to a hollow square (3000 ms) after a blank interval (200 ms). In order to control for visual-orthographic information between real characters and non-characters, we compared the related or unrelated conditions to the perceptual control condition for the fMRI analyses.
STIMULUS CHARACTERISTICS
Several lexical variables were controlled across the related and unrelated conditions. First, all Chinese characters were monosyllabic. Second, the fi rst character and the second character did not share radicals. Third, the fi rst character and the second character did not form a word (Wu and Liu, 1987;Sinica Corpus, 1998). Fourth, the number of nouns, verbs, adjectives, adverbs was similar for strong (48%, 23%, 29%, and 0%), weak (48%, 23%, 25%, and 4%) and unrelated pairs (50%, 23%, 21%, and 6%), based on their most frequent usage in Academia Sinica balanced corpus (Sinica Corpus, 1998). In addition, we split the related condition into strong and weak pairs, each pair with 24 trials, and calculated two word (fi rst, second) by three relatedness (strong, weak, unrelated) ANOVAs on stimulus characteristics. First, characters were matched for visual complexity (in terms of strokes per character) across conditions. The main effect of relatedness, F(2,138) = 0.68, p > 0.05, nor its interaction with word was signifi cant, F(2,138) = 2.85, p > 0.05. In addition, the correlation of visual complexity (fi rst or second words) with association strength was not signifi cant, r = −0.20, p > 0.05. Second, characters were matched for written frequency for adults (Wu and Liu, 1987) and written familiarity for children across conditions. For written frequency, the main effect of relatedness, F(2,138) = 1.00, p > 0.05, nor its interaction with word was signifi cant, F(2,138) = 2.41, p > 0.05. In addition, the correlation of frequency (fi rst or second words) with association strength was not signifi cant, r = −0.02, p > 0.05. Familiarity scores were obtained from pre-tests in which all the characters were rated on a 7-point scale by 30 age-matched children who were native Mandarin speakers from Taiwan. The instruction for written familiarity asked the children how often they saw the word in books, newspaper, and magazines. The 30 children were equally distributed across the 8-to 15-year-old age range. For written familiarity, the main effect of relatedness, F(2,138) = 0.7, p > 0.05, nor its interaction with word was signifi cant, F(2,138) = 2.62, p > 0.05. Finally, the semantic relation for related pairs (Lee et al., in press) was a dichotomous scale (categorical coding:1, functional coding:0). The point-biserial correlation of the measure of semantic relation with association strength was not signifi cant, r = −0.05, p > 0.05, indicating that association effects should not be due to semantic relation differences.
MRI DATA ACQUISITION
Participants lay in the scanner with their head position secured. An optical response box was placed in the participants' right hand. The (WISC-III) Chinese version (The Psychological Corporation, 1999). Participants' standard scores (mean ± SD) were 113 ± 10 on the verbal scale and 114 ± 11 on the performance scale.
FUNCTIONAL ACTIVATION TASKS
The children were given two practice sessions, one outside the scanner and the other in the scanner, to make sure that they understood the task. The practice items were different stimuli than those used in fMRI sessions. Each participant was at least 80% correct for each condition separately for both practice sessions. In the meaning judgment task, two visual Chinese characters were presented sequentially and the participant had to determine whether the character pair was related in meaning. Trials lasted 4500 ms and consisted of a solid square (500 ms), followed by the fi rst character (800 ms), a 200-ms blank interval, and the second character for 3000 ms. The duration of 800 ms for the fi rst word and a 200-ms ISI was the same as our previous English study (Chou et al., 2006a). The duration of 3 s for the second word was based on our previous Chinese study testing adults, healthy children, and children with smaller vocabulary sizes (Lee et al., in press). The upper bound of the mean + 2.5 SD of reaction time for children with smaller vocabulary sizes was close to 3 s. Thus, the duration of 3 s for the second word was chosen for a previous adult study (Chou et al., in press) and for this child study. The participant was instructed to make a response during the presentation of the second character. Forty-eight character pairs were semantically related according to their free association values (mean = 0.14, SD = 0.13, ranging from 0.73 to 0.01) (Hue et al., 2005). The Chinese norms (Hue et al., 2005) were created by presenting 100 native Mandarin-Chinese speakers with a list of target words and asking them to generate the fi rst word that came to mind. For example, if 40 out of the 100 participants generated the same word to a given target, the association strength was 0.4 for the word pair. This same procedure has been used in English (Nelson et al., 1998). Even though some of the word pairs had low association values in the related pairs, on average, the participants in our study were able to determine they were related. For example, the average accuracy for the word pair with association strength 0.01 was above 70%. Character pairs were arranged in a continuous variable according to association values. The distribution of association values was positively skewed so a logarithmic transformation was used (Howell, 2006). When values are near-zero, one can use log(X + 1) to perform a logarithmic transformation, but we used log(100X + 1) in order to get a positive number after transformation (mean = 1.04, SD = 0.36, ranging from 1.86 to 0.30). Such a logarithmic transformation has been shown to be suitable for item-level parametric analyses in developmental studies (Bolger et al., 2008). In addition, 24 word pairs were semantically unrelated with zero association values. The participants were instructed to quickly and accurately press with their right hand the yes button to the related pairs and the no button to the unrelated pairs. The perceptual control condition had 24 pairs of non-characters. Non-characters were created by replacing radicals of real characters with other radicals that did not form real Chinese characters. Noncharacters were larger (50 font size) than real characters (40 font size) in order to encourage participants to perform the task based on the recognition of low level visual similarity and not on the extraction of semantic information. The size was a cue for participants to judge head coil was positioned over the participants' head. Participants viewed visual stimuli projected onto a screen via a mirror attached to the inside of the head coil. This study adopted an event-related design. Each participant performed two functional runs. Each run took 4.7 min.
All images were acquired using a 3 Tesla Siemens scanner. Gradient-echo localizer images were acquired to determine the placement of the functional slices. For the functional imaging studies, a susceptibility weighted single-shot EPI (echo planar imaging) method with BOLD (blood oxygenation level-dependent) was used. Functional images were interleaved from bottom to top collected parallel to the AC-PC plane. The following scan parameters were used: TE = 24 ms, fl ip angle = 90°, matrix size = 64 × 64, fi eld of view = 25.6 cm, slice thickness = 3 mm, number of slices = 34; TR = 2000 ms. Each participant performed two 4.7-min functional runs. Each functional run had 4 dummy volumes discarded from fMRI analysis and 136 image volumes used for fMRI analysis. In addition, a high resolution, T1 weighted 3D image was acquired (TR = 1560 ms, TE = 3.68 ms, fl ip angle = 15°, matrix size = 256 × 256, fi eld of view = 25.6 cm, slice thickness = 1 mm, number of slices = 192). The orientation of the 3D image was identical to the functional slices. The task was administered in a pseudorandom order for all subjects, in which the order of related, unrelated, perceptual, and baseline trials was optimized for eventrelated design (Burock et al., 1998). We used the Optseq script for randomized event-related design (http://surfer.nmr.mgh.harvard. edu/optseq, written by D. Greve, Charlestown, MA, USA) that implemented Burock et al. (1998)'s approach.
IMAGE ANALYSIS
Data analysis was performed using SPM2 (Statistical Parametric Mapping). The functional images were corrected for differences in slice-acquisition time to the middle volume and were realigned to the fi rst volume in the scanning session using affi ne transformations. No participant had more than 3 mm of movement in any plane. We judged a 3-mm criterion to be appropriate because this was less than the in plane resolution of acquisition (4 mm). The average estimated motion over the whole run was small in all directions (x = 0.02 mm; y = 0.07 mm; z = 0.04 mm). In addition, the maximum displacement in any direction was small for each trial type (related = 0.019 mm, perceptual = 0.021 mm, baseline = 0.021 mm, unrelated = 0.021 mm). There were no signifi cant differences in maximum movement across trial types and there was not correlation of x, y or z movement with age. Co-registered images were normalized to the MNI (Montreal Neurological Institute) average template (12 linear affi ne parameters for brain size and position, 8 non-linear iterations and 2 × 2 × 2 nonlinear basis functions). We did not use a child template because given the age of our participants and the voxel size it was appropriate to use an adult template (Burgund et al., 2002). This also allowed for comparison of the results of the present study with other published adult studies. Statistical analyses were calculated on the smoothed data (10 mm isotropic Gaussian kernel), with a high pass fi lter (128 s cutoff period).
Data from each participant was entered into a general linear model using an event-related analysis procedure (Penny and Holmes, 2003). Character pairs were treated as individual events for analysis and modeled using a canonical HRF (Hemodynamic Response Function). There were four event types: related, unrelated, perceptual, and baseline. For the related pairs, association strength was an item-level parametric modulator in order to differentiate semantic relatedness as a continuous variable according to log transformed free association strength (Hue et al., 2005). The analysis of association strength was based on the related pairs and was not in comparison to baseline (also see Bolger et al., 2008). The resulting model coeffi cients for individual subjects were entered into subsequent second-order random-effects analyses in a whole brain analysis. All reported areas of activation were signifi cant using p < 0.05 corrected for FDR (false discovery rate) at the voxel level with a cluster size greater than or equal to 10 voxels.
Random-effects analysis using one-sample t-tests across all participants was used to determine whether activation during a contrast was signifi cant (i.e., parameter estimates were reliably greater than zero). First, we compared the related and unrelated pairs separately to the perceptual control condition, and the related to the unrelated pairs in a whole brain analysis. Second, we examined the effects of association strength for the related pairs, including reaction times as an item-based (within-subject) covariate. This allowed us to examine the association effects that were independent of reaction time differences. Positive effects indicated greater activation for related pairs with stronger association strength, whereas negative effects indicated greater activation for related pairs with weaker association strength. For the effects of association strength, we used the adult study (Chou et al., in press) to determine if a similar effect of association strength was present in children. An inclusive mask (p < 0.005 uncorrected) of left inferior parietal activation from the adult study was used to test for stronger association in this child study. An inclusive mask (p < 0.005 uncorrected) of left inferior frontal activation from the adult study was used to test for weaker association in this child study. Third, we used multiple regression to correlate the continuous variable of age in months with signal intensity for the related minus unrelated contrast, including accuracy or reaction time as a betweensubject covariate. This allowed us to examine age-related increases or decreases in activation that were independent of accuracy or reaction time differences. We extracted the beta values from the peak voxels of brain regions to visualize correlations for these age analyses.
BEHAVIORAL PERFORMANCE
Because 6% of accuracy values were located outside 2.5 SD from the group mean for each condition, trimming these outliers was applied to accuracy analysis. The subject-based accuracy (mean ± SD) for the related and unrelated conditions was 88 ± 10% and 96 ± 6%, respectively, with the related condition being less accurate than the unrelated condition, a paired t(32) = 4.31, p < 0.01. The subject-based reaction times (mean ± SD) measured from the onset of the second stimulus for the related and unrelated conditions were 1012 ± 244 ms and 992 ± 221 ms, respectively, with no signifi cant difference, a paired t(32) = 1.11, p = 0.28. The correlations of age with accuracy and reaction times were not signifi cant for the related pairs [r(33) = 0.216, p = 0.228; and r(33) = −0.338, p = 0.054, respectively]. Because character pairs were arranged in a continuous variable according to association strength, we calculated correlations between association strength for related pairs and behavioral performance. The item-based correlation between November 2009 | Volume 3 | Article 27 | 5 Chou et al.
Semantic development of Chinese characters accuracy and association strength was not signifi cant, r(48) = 0.27, p = 0.06; and the item-based correlation between reaction times and association strength was negative, r(48) = −0.39, p < 0.01. The subject-based accuracy and reaction times (mean ± SD) for the perceptual control were 99 ± 1% and 687 ± 152 ms, respectively. The subject-based accuracy and reaction times (mean ± SD) for the baseline were 99 ± 2% and 615 ± 155 ms, respectively.
BRAIN ACTIVATION PATTERNS
The presentation of the results will focus on brain regions that have been implicated in previous studies of semantic processing, namely left inferior frontal gyrus, posterior middle temporal gyrus, and inferior parietal lobule. All activation differences are reported in the tables. Because no signifi cant differences were found between the analysis of correct responses alone and the analysis that includes all responses, only results from the analysis with all responses are presented to equate the statistical power between conditions with different accuracies . This analysis was done by separately modeling correct and incorrect responses, and only examining condition differences for correct responses. We examined condition differences using correct responses for related versus perceptual, unrelated versus perceptual and related versus unrelated. The peak coordinates and corresponding Z values were similar for all responses and for correct responses only. Table 2 shows greater activation for the related or unrelated pairs compared to the perceptual control condition, and for the related compared to the unrelated pairs. Both related and unrelated pairs produced greater activation in left inferior frontal gyrus (IFG,BA 47,45) The effects of semantic association strength for the related pairs, partialing out the effect of reaction times in the scanner as a within-subject covariate, are shown in Table 3. Stronger association Note. See Table 2 note. The activation maps of stronger and weaker association (Chou et al., in press) were used as inclusive masks (p < 0.005 uncorrected) for the stronger and weaker association analyses in this study, respectively. with a task requiring association judgments as to whether character pairs were related in meaning. In order to more effectively measure activation within the semantic system, we manipulated the strength of association between the words in related pairs. Similar to previous child studies on English (Chou et al., 2006a,b), in Chinese stronger semantic association produced greater activation in left inferior parietal lobule (BA 39, 40), whereas weaker semantic association produced greater activation in the mid ventral region of left inferior frontal gyrus (BA 45). Regarding age effects on semantic processing, developmental increases in left posterior middle temporal gyrus were similar to those previously reported for English (BA 21) (Chou et al., 2006a,b;Szafl arski et al., 2006). However, developmental increases in left inferior parietal activation were more posterior in Chinese (BA 39) than in previous studies on English (BA 40) (Chou et al., 2006a), and left inferior frontal activation were more ventral in Chinese (BA 45, 47) than in previous studies on English (BA 6, 9) (Schapiro et al., 2004;Brown et al., 2005;Schmithorst et al., 2006;Szafl arski et al., 2006). After a discussion of the association strength effects, we will turn to a consideration of the developmental differences. Stronger semantic association produced greater activation in left inferior parietal lobule (BA 39,40). Activation in this region has previously been identifi ed in semantic association tasks in English children and Chinese adults (Chou et al., 2006a,b, in press). Greater activation in left inferior parietal lobule has been interpreted as evidence of semantic integration (Thompson et al., 2007). Left inferior parietal cortex has also been suggested to support the integration of lexical input into the larger units during semantic processing (Lau et al., 2008). Stronger association pairs may allow for greater integration because there are more overlapping features between the words or because the shared features are more characteristic of each of the words (Chou et al., 2006a,b). Greater integration for stronger association pairs may account for the increase in left inferior parietal lobule activation with increasing association strength in Chinese. Thus, the stronger association effect was similar between Chinese and English as well as between adults and children. produced greater activation in left inferior parietal lobule (BA 39, 40) (see Figure 1A). Weaker association produced greater activation in left mid ventral inferior frontal gyrus (BA 45) (see Figure 1B).
The correlations between age in months and activation, partialing out the effect of accuracy or reaction time in the scanner as a between-subject covariate, are shown in Table 4. Both correlations show that increasing age was correlated with greater activation for the related pairs compared to the unrelated pairs in the anterior and mid ventral regions of left inferior frontal gyrus (BA 45,47), left posterior middle temporal gyrus (BA 21), and left inferior parietal lobule (BA 39) (Figure 2). For descriptive purposes, we visualize the age effects by the correlations between age in months and beta values from the peak voxels of these three regions for the related pairs (correlations in IFG, MTG, and IPL were r = 0.44; r = 0.41; r = 0.43, respectively), as well as for the unrelated pairs (correlations in IFG, MTG, and IPL were r = 0.31; r = 0.05; r = 0.07, respectively). There were no negative age effects for the related minus unrelated pairs.
DISCUSSION
The neural correlates of semantic processing in children (8-to 15-year olds) to visually presented Chinese characters were examined We also found that weaker semantic association produced greater activation in the mid-ventral region of left inferior frontal gyrus (BA 45), similar to those reported in English children (Chou et al., 2006a) and Chinese adults (Chou et al., in press). Previous studies in English suggest that this region is involved in effortful semantic processing, particularly when there is increased demands on the process of selecting relevant semantic knowledge or when comparing words along semantic features (Thompson-Schill et al., 1997Fletcher et al., 1998;Whatmough et al., 2002;Blumenfeld et al., 2006). Badre and Wagner (2007) propose that the anterior ventral region of left inferior frontal gyrus (BA 47) may support controlled access to stored semantic representations, whereas the mid-ventral region of left inferior frontal gyrus (BA 45) may support increased selection demands to process active representations. Of particular relevance to the current study, semantic judgments to weaker association pairs produced greater activation in the mid-ventral region of left inferior frontal gyrus as compared to stronger association pairs. Greater activation for weaker association pairs could result from increased demands on the selection of appropriate semantic features in Chinese. Thus, the weaker association effect was similar between Chinese and English as well as between adults and children.
Developmental increases in left posterior middle temporal gyrus in Chinese are consistent with English studies showing age-related increases in this region (Chou et al., 2006a,b;Szafl arski et al., 2006). Previous child studies in English and Chinese implicate left posterior middle temporal gyrus in semantic processes (Chou et al., 2006a;Cao et al., 2009). Several studies suggest that the best candidate for the storage of lexical representations is in left posterior middle temporal gyrus Poeppel, 2004, 2007;Martin, 2007). Greater activation over age in this region may be associated with increasing elaboration of semantic representations, i.e. a greater number of semantic representations with more interconnections between these representations. Behavioral research shows that as vocabulary knowledge increases, the child's semantic system is gradually elaborated due to a greater number of conceptual links in Chinese and English (McGregor and Appel, 2002;Lee et al., in press). Therefore, the developmental increase in left posterior middle temporal gyrus may refl ect a general process that is universal across languages.
Developmental increases in left inferior parietal lobule [−40, −69, 34] in the present Chinese study are more posterior than developmental effects in this region [−45, −33, 40] For descriptive purposes, the graphs below are used to visualize the whole brain data that are shown above. The scatterplots of the correlation of age in months with the related (blue) and unrelated (red) conditions in these three regions are presented below the brain images. Beta values were taken from the peak voxel of the related minus unrelated contrast partialed for reaction time.
There are two potential limitations of the study. First, the related condition had twice as many trials as all other conditions, possibly resulting in higher signal-to-noise ratio in the related condition. We checked that in three critical regions (left inferior frontal gyrus, left middle temporal gyrus, and left inferior parietal lobule) signal was greater for the related than the unrelated condition. However, this difference should not have affected the main results of the paper as the strength of association effect was examined within the related trials, and age effects were examined for the related condition. Second, it is possible that the non-characters for the perceptual control condition could have been in part processed linguistically because radicals of real characters were replaced with other radicals to form non-characters. However, the comparison of the related or unrelated to the perceptual condition revealed activation in both left inferior frontal gyrus and left middle temporal gyrus, suggesting that real characters more robustly activated the semantic system than non-characters. Moreover, our association and age analyses examined only the related characters. Thus, the non-characters would be unlikely to have infl uenced the results of these analyses.
In conclusion, our study of Chinese children showed association strength effects in left inferior parietal lobule and in mid-ventral regions of left inferior frontal gyrus. These brain regions have been implicated in semantic integration and selection, respectively. We additionally showed that there were agerelated increases in left posterior middle temporal gyrus, similar to those reported in previous studies of English, suggesting that older children have more elaborated semantic representations across scripts. Because Chinese has a more consistent relationship in the mapping between orthography and semantics and a less consistent relationship in mapping between orthography and phonology, the developmental increases in the posterior region of left inferior parietal lobule and in the ventral regions of left inferior frontal gyrus suggests that reading acquisition relies more on the mapping from orthography to semantics in Chinese children at word level. studies of English using the same visual semantic task (Chou et al., 2006a). The anterior region of the left inferior parietal lobule has been implicated as part of the phonological loop (Paulesu et al., 1993) or the phonological store (Chen and Desmond, 2005), whereas the posterior region of left inferior parietal lobule has been suggested to support the integration of lexical input into larger units for semantic processing (Lau et al., 2008). Because reading for meaning places greater demands on the mapping from orthography to semantics at word level in Chinese (Leck et al., 1995;Zhou and Marslen-Wilson, 2000;Feng et al., 2001), developmental increases in the posterior region of left inferior parietal lobule may be related to more extensive semantic integration in older children. This suggestion is supported by our fi nding reported above that stronger association pairs, which can be more thoroughly integrated, also produced greater activation than weaker association pairs in the posterior region of left inferior parietal lobule. The developmental increases in the anterior region of left inferior parietal lobule in English may be due to greater involvement of phonological processing when reading in an alphabetic language.
In contrast to developmental studies of English, our study showed an age-related increase in ventral regions of left inferior frontal gyrus for Chinese children. Previous studies in English have shown developmental increases in more dorsal frontal regions in auditory narrative comprehension at [−42, 7, 30] and in verb generation in response to a noun at [−49, 3, 39] (Brown et al., 2005), at [−46, 7, 25] (Schapiro et al., 2004), and at [−47, 3, 30] (Szafl arski et al., 2006). Our coordinates in left inferior frontal gyrus at [−51, 27, 10] (BA 45) and at [−48, 21,−3] (BA 47) showing developmental effects in Chinese are substantially ventral to the coordinates reported in English studies. The subparts of left inferior frontal gyrus have been associated with distinct cognitive processes involved in language tasks. In general, the dorsal region of left inferior frontal gyrus is thought to be specialized for processing phonological representations, while the ventral region of left inferior frontal gyrus is proposed to be specialized for manipulating semantic representations (Poldrack et al., 1999), including controlled retrieval and selection mechanisms in ventral prefrontal regions (Badre, et al., 2005). Because reading for meaning places greater demands on mapping from orthography to semantics in Chinese (Leck et al., 1995;Zhou and Marslen-Wilson, 2000;Feng et al., 2001), older children may have learned to engage more thoroughly in controlled retrieval and selection of semantic knowledge at word level. The developmental increases in dorsal regions of left inferior frontal gyrus in English may refl ect greater phonological involvement in alphabetic languages in older children. | 2014-10-01T00:00:00.000Z | 2009-06-13T00:00:00.000 | {
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118607891 | pes2o/s2orc | v3-fos-license | Periodic waves in two-component Bose-Einstein condensates with repulsive interactions between atoms
We consider periodic waves in miscible two-component Bose-Einstein condensates with repulsive nonlinear interactions constants. Exact one-phase solution is found for the case when all these constants are equal to each other (i.e., for Manakov limit). New types of nonlinear polarization waves are considered in detail. The connection of the solutions found with experimentally observed periodic structures in two-component condensates is discussed.
Introduction. -Possibility of relative motion between two species in two-component Bose-Einstein condensates (BECs) leads to a rich phenomenology of nonlinear wave structures which can be generated in such systems. For example, in a two-component BEC with different values of intra-and inter-atomic interaction constants there exist two characteristic velocities of linear wavesthe sound velocity of density waves with in-phase motion of the two species and the polarization wave velocity with counter-phase motion of the two species (see, e.g., [1]). Correspondingly, there are two Mach cones and two channels of Cherenkov radiation of Bogoliubov linear waves by an obstacle moving with high enough speed [2]. Then interference of Cherenkov waves yields stationary ship wave patterns located outside the corresponding Mach cones. Besides that, a supersonic flow past an obstacle can generate oblique density solitons [3,4], half-solitons [5], or oblique polarization breathers [6]. These structures have been observed in experiments with flows of polariton condensates past obstacles [7][8][9]. In these experiments, both components had the same incident flow velocity and the wave pattern was created due to the action of the obstacle's potential. However, there exist other mechanisms of formation of nonlinear wave patterns. In particular, such patterns can be created by development of instability in the system, and such a modulation instability can be effective in the repulsive two-component condensates provided there exists large enough relative velocity between the components [10]. In the experiments [11][12][13] it (a) E-mail: kamch@isan.troitsk.ru was demonstrated that the relative motion between two species leads at the nonlinear stage of evolution to formation of the periodic polarization wave with locations of the crests in the density distribution of one component coinciding with locations of the troughs in the density distribution of the other component, so that the total density remains practically constant (although gradually changing along the trap at a larger scale of distance). These observations pose the problem of finding periodic solutions of the Gross-Pitaevskii (GP) equations describing dynamics of the two-component BECs. Although this problems was addressed in a number of papers (see, e.g., [14,15] and references therein), the general enough solution in the form convenient for applications is still absent. In this Letter, we shall present such a solution and discuss its possible applications to BEC dynamics.
General solution. -In accordance with the experiments [11][12][13], we consider a miscible two-component condensate confined in a one-dimensional trap which in the first approximation can be considered as a uniform cylinder. In the mean field approximation the dynamics of BEC can be described with a good accuracy by two-component GP equations p-1 where Ψ ± are wave functions of the condensate's components and the nonlinearity constants g ij = 4π 2 a ij /m can be expressed in terms of the scattering lengths a ij . We suppose that the components consist of atoms in two different electronic states and, hence, they have the same mass m. These components can mix if the constants of the nonlinear interaction satisfy the condition g 2 12 < g 11 g 22 (see [16,17]). In the experiments of refs. [11][12][13] these constants correspond to the states |1, −1 and |2, −2 of hyperfine structure of atoms 87 Rb with the scattering lengths equal to a 11 = 100.4a 0 , a 12 = 98.98a 0 and a 22 = 98.98a 0 , where a 0 is the Bohr radius [18]. Thus, they satisfy the above condition and, at the same time, they are very close to each other. Therefore for the description of the dynamics of the condensate we can accept that these constants have the same value g. It is worth noticing that in this case the velocity threshold for instability studied in ref. [10] vanishes, that is an arbitrarily small relative velocity between the components leads to growth of the polarization wave. In a rarefied enough condensate the transverse motion of atoms is described by the ground state function of a two-dimensional oscillator. Averaging over this state reduces the system (1) to the effectively one-dimensional system which describes the axial motion of the condensate's components along the coordinate x. Then the effective nonlinearity constant takes the value g 1D = g/(2πa 2 ⊥ ) where a ⊥ = ( /mω ⊥ ) 1/2 and ω ⊥ is the frequency of the transverse oscillations of atoms in the trap. It is convenient to introduce non-dimensional variables in the following way. Let ρ ch be a characteristic density of atoms, say, at the trap center. Then we take the healing length / √ mg 1D ρ ch as a unit of length, and the time /(g 1D ρ ch ) during which the healing length is traversed by a wave propagating with the sound velocity g 1D ρ ch /m as a unit of time. If we introduce also the wave functions ψ ± normalized according to Ψ ± = √ ρ ch ψ ± , then we arrive at the so-called Manakov system [19], where for convenience we use the previous notation for the non-dimensional variables x and t: It is convenient to represent a two-component order parameter (ψ + (x, t), ψ − (x, t)) as a spinor variable [20] Here ρ(x, t) = |ψ + | 2 + |ψ − | 2 denotes the total density of the condensate and Φ(x, t) has the meaning of the velocity potential of its in-phase motion; the angle θ(x, t) is the variable describing the relative density of the two components (cos θ = (|ψ + | 2 − |ψ − | 2 )/ρ) and φ(x, t) is the potential of their relative (counter-phase) motion. Accordingly, the densities of the components of the condensate are given by and their phases are defined as ϕ where U = Φ x and v = φ x . Substitution of eq. (3) into eq. (2) yields the system [21] We shall confine ourselves to situations when both components have equal chemical potentials µ and the wave propagates with velocity V . Hence, we look for the solution of the system (6) in the form In this case the system (6) can be integrated and we shall describe briefly the main steps of this calculation. Substitution of eqs. (7) into the first equation (6) and integration gives where A is an integration constant. The function φ ξ can be excluded from (8) with help of the last equation (6) where φ t = −V φ ξ . This gives the equation for Φ ξ ≡ Φ 0,ξ whose solution reads where The substitution of the expressions for Φ ξ and φ ξ into the third equation (6) gives equation for K, whose elementary integration yields where B is an integration constant. Equating this to the expression for K presented in (10), we get the equation which can be integrated once to give p-2 where C 2 is an integration constant (it is clear from eq. (12) that it must be positive). At last, the substitution of the obtained expressions for θ ξ , U = Φ ξ , v = φ ξ into the second equation (6) where Φ t = −2µ − V Φ ξ leads to the equation which again can be integrated once to give where and D is one more integration constant. It is remarkable that the variable ρ is separated in eq. (13) from the other variables. This is a consequence of the complete integrability [19] of the Manakov limit (2) of the two-component GP equations, although we have not used here this fact explicitly. The solution of eq. (13) is parameterized by three zeroes of the polyno- and it can be expressed in standard notation in terms of the Jacobi elliptic function, where m = (ρ 2 − ρ 1 )/(ρ 3 − ρ 1 ). In this solution, the total density ρ oscillates in the interval ρ 1 ≤ ρ ≤ ρ 2 and, according to its physical meaning, ρ must be positive; hence all ρ i , i = 1, 2, 3, are positive, too. Their product denoted as R 2 ≡ ρ 1 ρ 2 ρ 3 equals to R 2 = A 2 + C 2 . Therefore, we can introduce, instead of the constants A, B, C, more convenient parameters β and γ as follows, The last definition implies that |B| ≤ R = √ ρ 1 ρ 2 ρ 3 what follows from the observation that according to eq. (12) the angle θ oscillates between the values θ 1 , θ 2 determined by the condition that the right-hand side of eq. (12) vanishes, that is by the equation B = A cos θ 1,2 ± C sin θ 1,2 = R cos(γ ∓ θ 1,2 ) which gives |B| ≤ R. We define and suppose for definiteness that the parameters β and γ are chosen in such a way that cos θ 1 ≤ cos θ 2 . Then eq. (12) reduces to Its integration yields the solution for θ(ξ): cos θ(ξ) = cos θ 1 sin 2 X(ξ) 2 + cos θ 2 cos 2 X(ξ) 2 , where X 0 is an integration constant. The integral in (20) can be expressed in terms of Weierstrass elliptic functions (see, e.g., [22]), however it is more convenient for future study to keep it in a non-integrated form. Equations (15) and (19), (20) determine the fields ρ(x, t) and θ(x, t). Their substitution into eqs. (9) and (5) yields the flow velocities of the BEC components: (21) Subsequent integration of these formulae and account of the expression for the chemical potential gives the expressions for the phases ϕ ± (see eqs. (5)). This completes the derivation of the periodic solution of the system (2). It is parameterized by six constant parameters V, ρ 1 , ρ 2 , ρ 3 , β, γ.
It is important to notice that only the total density ρ(x, t) is a periodic function of ξ = x − V t; the angle θ is a periodic function of X which is a quite complicated function of ξ (see eq. (20)). Therefore the densities ρ ± of the components and their velocities v ± vary with x and t in a complicated way. However, the situation greatly simplifies in important particular cases discussed below.
Nonlinear polarization wave. -The spinor (3) can be characterized by the polarization vector S = χ † σχ (here σ = (σ 1 , σ 2 , σ 3 ) is a vector of Pauli matrices) with the components S = (sin θ cos φ, sin θ sin φ, cos θ). Hence, the relative motion of two components of BEC can be represented as a polarization dynamics. Here we shall consider the above solution in the case of pure polarization dynamics when the total density is constant. This takes place for ρ 1 = ρ 2 ≡ ρ 0 , when R = ρ 0 √ ρ 3 and ρ(ξ) ≡ ρ 0 . Hence we get This is a linear function of x and t, therefore such a polarization wave represented by eq. (19) depends periodically on the space and time variables. Instead of the parameter ρ 3 , it is convenient to introduce other parameters with clearer physical meaning. Let us define mean fluxes of the components averaged over either x or t: j ± = ρ ± v ± . A simple calculation gives Obviously, the amplitude of this wave can be measured by the parameter (θ 2 − θ 1 )/2 = γ. First, we shall consider linear waves propagating over a quiescent background with γ = 0 and j ± = 0. The last condition is satisfied if √ ρ 3 = V . Then in the small amplitude limit γ ≪ 1 eq. (19) takes the form p-3 Here the wave number and the frequency are equal, respectively, to k = 2V , ω = 2V 2 , and they satisfy the dispersion relation This formula suggests that, on the contrary to Bogoliubov density waves, the linear polarization waves can have arbitrarily small velocities what is confirmed by the direct analysis of the linearized two-component GP equationsthe "sound" velocity of the polarization waves vanishes in the Manakov limit (see, e.g., [21]). Hence, the parameter ρ 3 = V 2 is not limited here by the inequality ρ 3 > ρ 0 implied above which is an artefact of our method of derivation based on the study of waves with changing total density.
In a nonlinear wave both fluxes are equal to zero if either cos β = 0 or √ ρ 3 = V . The first case corresponds to the condensates with equal mean densities of the two components. In this case we get the nonlinear wave with where the wave number and the dispersion relation depend on the amplitude γ: The second opportunity This means that if j 0 > 0, then β < π/2, and vice versa. The wave number and the dispersion relation are given by We can obtain a stationary wave with V = 0 if j 2 + = j 2 − . In this case eqs. (24) yield Suppose that this wave is excited from a quiescent uniform state with ratio of the components densities ρ − /ρ + = tan 2 (β 0 /2). If this parameter remains constant during the excitation process, then the parameters in the excited wave satisfy the condition cos β cos γ = cos β 0 .
Hence, the wave number of the stationary wave equals to Quasi-soliton wave. -Now we shall turn to the soliton limit of the solution (15) for the total density when ρ 2 = ρ 3 = ρ 0 and this solution takes the form (to simplify the notation, we suppose that the soliton is located in our reference frame at the point x = V t). Far enough from the soliton's center, the total density becomes uniform and equal to ρ ∼ = ρ 0 . However, for θ 1 = θ 2 , the components densities oscillate here and compose the polarization wave discussed above. The variable X(x, t) defined by eq. (20) can be expressed now in terms of elementary functions, (33) (for simplicity, we omitted here X 0 ). It is clear that at |ξ| → ∞ the variable X becomes a linear function of ξ. If we demand that at infinity both fluxes j ± are equal to zero, then we obtain Thus, this quasi-soliton wave transforms here into "slow" polarization waves with the parameter ρ 1 < ρ 0 playing the role of ρ 3 in the formulae of the preceding section. (This confirms our statement above that the parameters in the polarization wave are not limited by the condition ρ 3 > ρ 0 .) We call this solution a quasi-soliton because it is not localized in space on the contrary to the usual soliton solutions of nonlinear wave equations; rather, generally speaking, it represents a "defect" in the polarization wave. This defect manifests itself as a dip in the distribution of the total density in the form of a dark soliton. The whole structure propagates with velocity V . Equation (34) can be rewritten in the form where V s = √ ρ 1 is the soliton's velocity related with the minimal density ρ 1 at its center in the case of a onecomponent condensate. Thus, a quasi-soliton propagates slower than a usual dark soliton with the same depth. When the amplitude of the polarization oscillations vanishes, γ = 0, then the quasi-soliton transforms into a wellknown localized dark-dark soliton with constant ratio of the components densities (see, e.g., [4]).
The distributions of the total density and the components densities in the quasi-soliton solution are illustrated in fig. 1. It is clearly seen that far enough from the soliton center the densities oscillate in counter-phase resulting in the constant total density. The distributions of the flow velocities in this wave are shown in fig. 2. They show that the relative motion between the components is crucially important for formation of such a wave.
Discussion. -One can suppose that this type of a polarization wave has been observed in the experiments [11][12][13]. In refs. [11,12] the relative motion between the components of the two-state 87 Rb condensate was induced by a gradient of an external magnetic field applied along the axial direction of a cigar-shaped optical dipole trap. In [11], apparently a dispersive shock wave represented by a modulated polarization wave has been observed with large-amplitude oscillations of the polarization and very small oscillations of the total density. In [12] a dense lattice of the polarization oscillations has been created by a counterflow through the condensate due to the modulational instability of a uniform state [10]. We believe that the polarization waves, whose theory has been developed here, can be formed at a nonlinear stage of evolution of the modulational instability. To support this supposition, we shall make here a rough theoretical estimate of the parameters of the wave and compare it with the experimental data.
The main qualitative result of the theory is the statement that the wave number of the polarization wave is proportional to the velocity of the wave, k ∝ V , where the proportionality coefficient depends on the amplitude of the wave (see, e.g., (27) or (29)). The dependence of k on the parameters β and γ is weak; one can find that it introduces an essential correction (a factor about 2) if only the density of one of the components is less than 20% of the density of the other component. To transform the approximate relation (λ is the wavelength) to a dimensional form, we calculate the healing length ξ 1D = / √ mg 1D ρ ch ∼ = 2.4 × 10 −5 cm and the sound velocity c s = g 1D ρ ch /m ∼ = 0.3 cm · s −1 . As a result we get the formula From the data presented in [11] one can find that the velocity of the polarization wave equals to V ∼ = 1.8 × p-5 10 −2 cm · s −1 . Substitution of these experimental parameters into eq. (36) gives λ ≈ 13 µm. According to the experimental plots presented in [11], the experimental value of the wavelength equals to λ ∼ = 15−18 µm. Thus, taking into account rough approximations which we have made in our estimate, we can consider this as a satisfactory agreement of the theory with the experiment. For more accurate theoretical description of formation of the dispersive shock wave generated in the experiment, one needs to develop the theory of time-dependent modulations (Whitham theory) which can be the subject of a separate study.
Conclusion. -We have found an exact analytical periodic solution of the GP equations in the Manakov limit corresponding to equal constants of repulsive interactions between atoms. This model can be considered as an approximation to description of dynamics of a "weakly miscible" two-component condensate [11,12,23]. In immiscible two-component condensates other types of solitons generated by counterflow between the two components are possible [24]. The solution found here includes as limiting cases the nonlinear polarization waves and quasi-solitons which can be thought of as defects in the polarization waves. In the limit of vanishing polarization oscillations, a quasi-soliton transforms to a standard dark Manakov soliton. Existence of new solutions of the Manakov system, which can be considered as generalizations of onecomponent dark solitons, poses the important problem of their stability. One may hope that the methods of previous studies (see, e.g., [25,26]) can be generalized to this new situation, although the problem of stability of polarization waves and quasi-solitons is far beyond the scope of this Letter. We indicate only that possible relation of the polarization waves with experimentally observed wave patterns suggests their stability.
In conclusion we remark that the theory developed here can be also applied to description of the polarization dynamics of light pulses in nonlinear optical systems and the method used here can be extended to other choices of signs of the nonlinear interaction constants. | 2013-10-07T15:26:28.000Z | 2013-08-10T00:00:00.000 | {
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209418477 | pes2o/s2orc | v3-fos-license | Population pharmacokinetic modelling of busulfan and the influence of body composition in paediatric Fanconi anaemia patients
Aims Fanconi anaemia (FA) is a rare disorder characterized by progressive bone marrow failure that requires haematopoietic cell transplantation (HCT). Busulfan is used in conditioning regimens prior to HCT. Doses used in non‐FA patients cause life‐threatening toxicities in FA patients and data on busulfan pharmacokinetics (PK) in this population are limited. This study characterized busulfan PK in paediatric FA patients using population PK modelling and evaluated the effect of body composition on steady‐state concentrations (C ss). Methods A total of 200 busulfan plasma concentrations in 29 FA patients from a recent study (http://Clinicaltrials.gov; NCT01082133) were available for population PK modelling. The effect of different body size‐scaled doses and body compositions on C ss was investigated using population PK modelling. Results Fat free mass (FFM) was identified as the best size descriptor in a two‐compartment busulfan PK model in FA patients. Conventional dosing, based on an amount of busulfan per kilogram of total body mass, resulted in higher C ss in FA patients with higher body mass index (BMI). A newly proposed FFM‐based dosing strategy would eliminate the observed trend of higher concentrations in high BMI patients, and achieve consistent C ss across a wide BMI spectrum. Conclusions This is the first study to describe the population PK of busulfan in paediatric FA patients. The proposed model will facilitate PK model‐informed precision dosing. FFM‐based dosing is expected to improve the probability of achieving target C ss, particularly in obese patients, while minimizing the risk of overdosing.
| INTRODUCTION
Fanconi anaemia (FA) is a rare inherited genetically and phenotypically heterogeneous disorder that causes progressive bone marrow failure. 1 FA patients are predisposed to developing leukaemia and solid tumours. Median life expectancy is therefore 23 years. 2 Hence, it affects mostly paediatric patients. Various congenital malformations are often seen, including short stature and kidney abnormalities associated with decreased renal function. 2 These anatomical and physiological characteristics might influence drug behaviour in FA patients, therefore understanding the differences in drug disposition compared to non-FA patients is of clinical importance.
Allogeneic haematopoietic cell transplantation (HCT) is an effective therapy to treat the haematological complications of patients with FA. 3 Busulfan is widely used in conditioning regimens prior to HCT for various haematological disorders. Due to large variability in busulfan pharmacokinetics (PK) in combination with its narrow therapeutic index, monitoring of busulfan concentration is recommended to maximize the effect while minimizing the incidence of side effects. High systemic exposure to busulfan is correlated with toxicities such as sinusoidal obstruction syndrome and acute graft-versus-host disease in adult patients undergoing HCT. 4,5 In contrast, low exposure is associated with an increased risk of graft rejection and relapse. 6,7 Toxicities emerging from conditioning regimens are more common in FA patients due to their high chemotherapy sensitivity, which is related to an inability to repair DNA damage. 8,9 Finding the right dose in FA patients has been a challenge for clinicians, in part due to limited information on the PK of busulfan in this patient population.
Busulfan PK has been recently described in FA patients. 10 The aim of this study was to characterize the population PK of busulfan in paediatric patients with FA. The effects of differences in body size on busulfan PK in FA patients were specifically examined, since body weight has been reported to be an important predictive covariate in non-FA patients [10][11][12][13][14][15][16][17][18][19] and the FA patient population shows a unique physical characteristic with wide body weight distribution. That is, in general FA patients are of notably short stature and underweight compared to a relatively healthy paediatric population, 2 but overweight and obesity can still occur.
| Patients and study design
Data from a subset of patients who participated in a recent phase 2 study (ClinicalTrials.gov: NCT01082133) by Mehta et al. were used for the development of the population PK model. 20 The Institutional Review Boards of Cincinnati Children's Hospital Medical Center approved the study and all parents/guardians and/or patients provided written informed consent and assent before enrolment in the study. In brief, in the clinical study, two groups of patients received either an initial busulfan dose of 0.8 or 1 mg kg −1 (based on patient age and weight as previously reported 11,13 ), or a reduced dose of 0.6 or 0.8 mg kg −1 as a 2-hour intravenous infusion. Blood samples were collected at 0, 15 and 30 minutes, and 1.5, 2, 3 and 4 hours after the end of intravenous infusion of busulfan. Based on the PK results, subsequent busulfan doses were reduced (if necessary). 20 A total of 200 busulfan plasma concentrations from 29 FA patients after first dose were available for PK analysis. Busulfan concentrations were measured by gas chromatography with mass spectrometry detection as previously described. 21 The within-and between-day coefficients of variation for the assay were below 8%. The dynamic range of the assay was from 125 to 7500 ng mL −1 with a lower limit of quantification of 125 ng mL −1 . Patients' demographic data and clinical laboratory test results were collected as part of routine clinical care. was applied for all runs. Both one and two-compartment models were explored, and model selection was based on goodness-of-fit diagnostics plots, comparisons based on the minimum objective function value (OFV) and evaluation of the estimates of population fixed and random effect parameters. Interindividual variability was assessed using the following model (Equation (1)):
| Population PK modelling
What is already known about this subject • Busulfan has a narrow therapeutic index and is used in preparative regimens prior to haematopoietic cell transplantation in the rare disorder Fanconi anaemia (FA).
• Total body mass-based dosing is currently recommended in patients with FA.
• Body size is a predictive covariate for busulfan pharmacokinetics (PK) in non-FA paediatric patients.
What this study adds
• This is the first study describing population PK of busulfan in paediatric FA patients.
• Size-normalized busulfan PK in FA patients was similar to that as in non-FA patients.
• Fat free mass-based dosing is expected to improve target steady-state concentration attainment, particularly in obese patients, which would reduce variability in clinical outcomes.
where θ i is the estimated parameter value for individual i, θ TV is the typical value of parameter θ in the structural model, and η i is an interindividual random effect for individual i, which was assumed to follow a normal distribution with a mean of 0 and a variance of ω 2 . Investigated PK parameters were clearance (CL), volume of distribution of the central compartment (V 1 ), intercompartmental clearance (Q), and volume of distribution of the peripheral compartment (V 2 ). A proportional error model and a combined proportional and additive error model were explored to describe the residual error.
Both allometric and linear scaling were examined to account for differences in body size and composition as follows (Equation (2)): In this study, NFM was also assessed as a size descriptor according to previous reports. 17 FFM was used to calculate NFM as follows (Equation (5)): where Ffat is a fraction that reflects the role of fat (TBM minus FFM) on metabolic processes or physiological volumes. 22 If Ffat is 0, size is completely described by FFM, whereas if Ffat is 1, TBM describes the body size mass entirely.
Lastly, IBM was tested as a size descriptor, which has been suggested as a dosing mass index for several drugs in obese children. 26 IBM was calculated as follows (Equation (6)): where IBM is in kilograms and HT i is the height of individual i in centimetres (cm). 27 If the height of the patient exceeded 5 feet (154 cm), the IBM for males and females was calculated using the following formulas (Equation (7) and (8), respectively) 28 : PK. The differences in OFVs from nested models was assumed to be χ 2 distributed. A drop of more than 6.63 points (P < 0.01) was considered to be significant.
| Model evaluation
To evaluate the models and to identify potential bias due to model misspecification, the following diagnostic plots were used: observed value (DV) vs population-predicted value (PRED), DV vs individualpredicted value (IPRED), conditional weighted residuals (CWRES) vs PRED and CWRES vs time after start of the infusion. Non-parametric bootstrap analysis was run with 1000 resampled datasets. 29 The estimated medians and 95% confidence intervals were compared with the final model estimates. For the validation of the final model, the prediction-corrected visual predictive check (pcVPC) was used by simulating 1000 datasets. 30 The observations were compared with the distribution of the simulated concentrations.
| Individual steady-state concentration estimates by Bayesian estimation
Patient demographics, dosing information and busulfan plasma concentration-time data were used to estimate individual PK parameters by using the MwPharm++ software (version 1.5.1, MEDIWARE, Inc., Prague, Czech Republic), a therapeutic drug management application. In the application, the final population PK model parameters were used as prior population information for Bayesian estimation using individually observed concentrations as feedback. The workflow of this analysis is depicted in Figure S1. In this study, busulfan exposure was reflected as concentration at steady-state (C ss ). C ss was calculated using the following formula (Equation (9)) 31 : where AUC 0-∞ is the area under the curve from t = 0 to infinity and τ is the dosing interval.
| Statistical analysis
The correlation between a patient's BMI and the estimated busulfan C ss was assessed based on linear regression analysis using Gra-phPad Prism (version 7.0, GraphPad Software, Inc., San Diego, CA, USA). Differences in estimated C ss achieved between patient subpopulations were analysed by the nonparametric Mann-Whitney Utest.
| Patients
The demographic characteristics of the patients with FA are summarized in Table 1. Frequency histograms of the data are presented in 32 This study population included one overweight and four patients in the obese group. In comparison to the National Health and Nutrition Examination Survey (NHANES) data, 33 patients tended to have a short stature and be underweight, with a BMI on the lower end of the spectrum in both males and females (Figure 2).
| Population PK modelling
A two-compartment model with zero-order infusion adequately Table 2.
| DISCUSSION
This is the first study to characterize PK of busulfan in paediatric patients with FA through population PK analysis. Individual busulfan PK profiles were predicted adequately in FA patients by the population PK model, which was developed using FFM as the most impor- Patient's gender and several clinical laboratory test results were applied to clearance as potential covariates; however, they did not show any significant improvement of the model fit, which is consistent with other reports in non-FA patients. 14,15 Regarding the clearance of busulfan, the primary pathway is conjugation with glutathione in the liver by glutathione S-transferase (GST), with the GSTA1-1 isoform being the most active form of GST. 35 As hepatic conjugation capacity decreases in case of liver injury, this could affect the elimination of busulfan. Liver transaminases, ALT and AST, were elevated (ie, above the 97th percentile of the healthy paediatric reference value for that sex and age 36 ) in around half of the subjects, indicating potential liver damage in these patients. Yet these enzymes were not found to be significant covariates, which might indicate that liver damage in terms of ALT or AST elevation does not essentially affect the conjugation of busulfan in our population. However, no conclusions can be drawn due to the small sample size and the relatively small range of transaminase values.
Age has been found to be a significant covariate associated with busulfan PK in non-FA paediatric patients, 16,17,19 while this was not observed in our analysis. This might be explained by the difference in age in the study cohort, since the youngest patient in our study was 4.3 years of age, while other trials included patients as young as 0.1 years. Size-standardized busulfan clearance reaches 95% of the adult value at 2.5 year of age. 17 Hence, this could explain why no age effect was observed in our study cohort.
In this study, multiple body mass descriptors were examined to assess the effects of differences in body size on busulfan PK in paediatric FA patients. FFM proved to be the most informative predictive covariate and was implemented in the population PK model. [13][14][15][16][17] All values, which were often expressed corresponding to the median weight of the population, were allometrically scaled to a total body weight of 70 kg, using the theoretical power exponents of 0.75 and 1.0 for clearance and volume, respectively. Both one-and twocompartment models have been reported. Since one-compartment models inherently include only one volume of distribution (V d ), this value was compared to the sum of the estimated volumes for the central and peripheral compartment (V ss ) in two-compartment models. Black circles represent the estimated value, with whiskers showing the 95% confidence interval of the mean estimate, unless stated otherwise. † Whiskers show the 90% confidence interval received the same 0.6 mg kg −1 TBM dose. Still, obese patients were predicted to be exposed to higher concentrations of busulfan than non-obese patients (P = 0.0091). This could be explained by the tendency of busulfan to distribute over total body water. 38 Obese patients have more adipose tissue that is unlikely to contribute to the volume of distribution of busulfan due to its hydrophilic properties (ie, log P = −0.36 39
| CONCLUSION
A population PK model of busulfan in paediatric FA patients that incorporates FFM was developed that will allow improved prediction of busulfan exposure. FA patients were found to have similar sizenormalized busulfan PK as non-FA patients. Conventional dosing, which is based on an amount of busulfan per kg TBM, attains a higher C ss if the patient is more overweight, increasing the risk of overdosing.
Dosing based on FFM is expected to improve target exposure, especially in obese patients, since the achieved C ss is predicted to be unbiased over a wide range of BMI values.
ACKNOWLEGDEMENTS
We gratefully acknowledge the contributions of Nieko Punt, MSc, for guiding the development of the pharmacokinetic model of busulfan in FA patients. In addition, the insights provided by Min Dong, PhD, comparing the FA demographic data to the NHANES data, were essential for the evaluation of our data. | 2019-12-20T14:04:25.825Z | 2019-12-18T00:00:00.000 | {
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236659174 | pes2o/s2orc | v3-fos-license | Collagen I Induces Preeclampsia-Like Symptoms by Suppressing Proliferation and Invasion of Trophoblasts
Preeclampsia is a common obstetric disorder affecting 2-8% of pregnancy worldwide. Fibrosis is an important histological change occurring in preeclamptic placenta, and might depend on the excess deposition of collagen I. However, the role of fibrotic placenta and collagen I in the pathogenesis of preeclampsia remains unclear. Therefore, we analyzed the collagen deposition and the expression of Collagen I in human placenta by Masson staining, Sirius red staining and western blotting. Further, the role of collagen I in preeclampsia pathogenesis was studied in C57BL/6 mice. HTR-8/SVneo cells were used to investigate the mechanisms underlying the effects of collagen I in trophoblasts by transcriptome sequencing and pharmacological agonists. Human preeclamptic placenta exhibited a significantly higher degree of fibrosis in stem villi and terminal villi than normal placenta, and was characterized by collagen I deposition. In vivo, a single injection of collagen I on gestational day 0.5 led to an increase in systolic pressure of pregnant mice from gestational days 4.5–17.5, to a decrease in weight and number of embryos, and to enhanced placental collagen I expression and degree of fibrosis compared with control mice. In vitro, collagen I attenuated the proliferation and invasion of HTR-8SV/neo cells. This effect could be reversed by treatment with agonists of ERK and β-catenin. Moreover, transcriptome sequencing demonstrated that signaling pathways related to cell proliferation and invasion were significantly downregulated in HTR-8SV/neo cells. Thus, we propose that collagen I induced preeclampsia-like symptoms by suppressing the proliferation and invasion of trophoblasts through inhibition of the ERK phosphorylation and WNT/β-catenin signaling pathways. Our findings could pave the way to the discovery of small-molecule inhibitors for preeclampsia treatment and future studies with larger sample size are required.
Sirius red staining
After fixation in 4% paraformaldehyde of placenta, the slices were embedded in paraffin. Section of 3μm were stained by structural identification, with Masson's trichrome staining for collagen fiber observation, and with Sirius red stain for collagen identification.
Masson-trichrome staining
For Masson trichrome staining, the sections were stained using a Masson's trichrome staining kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. The slides were incubated in Weigert's iron hematoxylin (5 min), Biebrich Scarlet-Acid Fuchsin Solution (15 min), Phosphomolybdic-Acid Solution (15 min) and Aniline Blue Solution (10 min), all at room temperature. The slides were visualized under the light microscope. Collagen fiber were stained blue, while cytoplasm and red blood cells were stained red and nucleus blue and brown.
Fibrosis area% was calculated in µm digitally using the software NDP.view2 (Hamamatsu Corp, Japan). The area of tubulointerstitial fibrosis was measured in 5 random fields under ×200 magnification.
Sirius red staining
For Sirius Red staining, the Picrosirius Red stain kit (Abcam, Cambridge, UK) was utilized. Sections were stained with Weigert's iron Sumu essence dye for 15 min, rinsed for 5 min, and then washed with distilled water. The sections were covered with 200 μl Sirius red dye for 1 h. Each analyzed field was chosen randomly and the positive red-stained areas and red-yellow density were quantified using computerized image analysis software (NIH, MD, USA).
Western blotting
Placenta tissue and in vitro-treated cells was homogenized and protein extracted as described below.
Homogenized by a Qiacube machine (Qiagen, Courtaboeuf, Fance ) in RIPA lysis buffer (Thermo, Rockford, USA) on ice. The supernatants were collected after centrifugation at 12000×g at 4°C for 20 min. Protein concentration was determined using a BCA protein assay kit (Thermo, Rockford, USA), and whole lysates were mixed with 5×SDS loading buffer (Coolaber, Shanghai,China) at a ratio of 1:4. Protein samples were heated at 98°C for 5 min and were separated on SDS-polyacrylamide gels(Biodragon,Guangzhou,China). The separated proteins were then transferred to a PVDF membrane (Dogesce, Shanghai, China). The membrane blots were first probed with a primary antibody. After incubation with horseradish peroxidase-conjugated second antibody, autoradiograms were prepared using the enhanced chemiluminescent system to visualize the protein antigen. The signals were recorded using SYNGENE BIO IAMGING (GENE GNOME, Shanghai,China). Primary antibodies for Western Blot are rat anti-collagen I, anti-MMP9, anti-, anti-vimentin, anti-E-cadherin, anti-Ncadherin, anti-β -catenin, anti-ERK, anti-p-ERK and rabbit anti-GAPDH (Cell Signaling, San Jose, CA, USA). GAPDH was used as a protein loading control. The secondary antibody was HRP-conjugated anti-rabbit (Cell Signaling, San Jose, CA, USA). Images shown in the figures were representative of 5 individuals. ImageJ software (https://imagej.nih.gov/ij) was used for image acquisition and densitometric analysis of the gels.
Quantitative PCR (RT-qPCR)
Total RNA was extracted with Trizol reagent (Invitrogen, Corporation, USA) according to the manufacturer's instructions. The reverse transcription reaction was carried out with reverse transcription enzyme (Toyobo, Shanghai, China). Quantitative real-time PCR was carried out on an LightCycler96 real-time PCR system (Roche,Basel, Switzerland) and the specific primers for quantitative PCR are shown below (IGE,Guangzhou,China).
CCK-8
HTR-8/SVneo cells were seeded in 96-well plates at the density of 10000 cells per well in 250μl of complete culture medium. After treatments, three methods were utilized for cell proliferation analysis. Cell Counting Kit-8 (Beyotime, Guangzhou, China) analysis: 10 ul of CCK-8 was added to each well. The culture plates were shaken for 90 min and the optical density (OD) values were read at 450 nm.
Cell cycle
After treatment of collagen I, HTR-8SV/neo cells were harvested by trypsinization and library quality was assessed on the Agilent Bioanalyzer 2100 system.
Clustering and sequencing
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, CA, USA) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. | 2021-08-03T00:05:04.217Z | 2021-02-11T00:00:00.000 | {
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220883898 | pes2o/s2orc | v3-fos-license | An Educational Evaluation of a Journal Club Approach to Teaching Undergraduate Health Care Research
Background: Health care research is a common undergraduate health sciences requirement. There is limited literature regarding course structure, content, or learning outcomes; most courses have traditionally been taught through didactic lecture. This is misaligned with Generation Y learner values, as they desire guided learning, real-world examples, active engagement, learning through doing, and psychological safety. Methods: A “journal club” approach to teaching health care research was implemented at Northeastern University in Fall 2018. Each session involved (1) a moment of reflection; (2) an introduction to the topic; (3) 1 student methods report presentation; (4) 2 student “journal club” self-directed structured article summary presentations; (5) large-group discussion; (6) plus/delta feedback to instructor. Each student completed 2 “journal club” and 1 methods presentations, 6 peer reviews, CITI research training, a quality improvement survey, and a final course reflection. We utilized a convergent mixed-methods educational evaluation, integrating data from 3 distinct sources—a quality improvement survey, final student course reflections, and Plus/Delta feedback—which were analyzed via thematic analysis. The Northeastern University Institutional Review Board exempted the study. Results: Students appreciated the course structure and reported confidence in their critical appraisal abilities. Four qualitative themes emerged: (1) enabled a high degree of growth as students and scholars; (2) designed in thoughtful and unique format; (3) initially intimidated students and was academically challenging; and (4) prioritized and enabled psychological safety. Conclusions: Although initially intimidating and admittedly challenging, undergraduate health sciences students applauded the course’s curricular design and enabling of psychological safety, which aligned with Generation Y learner values, ultimately leading to growth in perceived and realized confidence and ability to critically review research articles.
Journal of Medical Education and Curricular Development
Moreover, generation Y students-born after 1980-prefer more active learning, often including instructor guidance, active engagement with others, learning through doing, and course material related to real-world experiences. 2,18,19 They also desire a strong classroom structure with consistent psychological safety. 2,18,20 We developed a convergent mixed-methods educational evaluation for a journal club approach to teaching undergraduate health care research to Generation Y students, complemented by multiple key educational design strategies to support deep learning. We add to the literature by illuminating students' perspectives on this approach, detailing their perceived educational impact on their understanding of and confidence with research methods via the use of a quality improvement survey, a final course reflection, and Plus/Delta feedback.
Setting and participants
All participating students were upper-level students in the Health Science major (550 total students) at the Bouvé College of Health Sciences (2000 total students) at Northeastern University in Boston, MA. In Fall of 2018, the senior authora medical and health professions educator-taught 2 sections of Health Care Research at Northeastern University. The morning course had 16 students while the afternoon course had 24 students (total n = 39). All classrooms contained a computer with a ceiling mounted projector and screen that enabled PowerPoint presentations, as well as a chalkboard.
Curriculum design
Kern's 6-step curriculum development process guided curriculum development. 21 Initially, the first author reviewed prior literature regarding how health care research is taught and spoke with other Northeastern University faculty members about their perceptions of what the students needed to be successful.
The instructor embedded multiple core cognitive science and adult learning principles in the course design, as outlined below. [21][22][23] First, students were consistently informed and reminded that research methods would be essential for their future careers as health professionals, and each course session included a "who is this?" component to introduce students to practicing health care researchers. Second, although journal club assignments were scaffolded by the instructor who provided a clear template for students, significant self-directed learning and autonomy among students was required. 15 Third, the instructor encouraged students to draw on their prior learning experiences in research and statistics. Fourth, readiness to learn was supported by the use of articles of increasing complexity throughout the course so that students had to continually revisit complex course concepts. Fifth, students' motivation to learn was encouraged through the development and maintenance of a "safe learning space," in which students consistently asked questions as well as provided and received feedback from the instructor and engaged in peer review. The intention was to cultivate a desire among all students to complete assignments at the highest level of their ability. Sixth, psychological safety was additionally encouraged via the use of a minute at the beginning of class to hit pause and "situate ourselves in our learning space" to remind students to actively choose to be engaged in their own learning for the session. 23 Finally, the journal club design enabled active learning, largely through large group discussion with student-led questions and supported spaced repetition through the interleaving of main concepts.
Course structure
The courses spanned 14 weeks, with 2 sessions per week at 140 minutes, amounting to 23 course sessions. Students were assigned a research article, and utilized the "self-directed structured summary" template to develop a 1-page summary. 15 This summary included the introduction, methods, results, and the conclusion, highlighting challenges or concerns students noted (Appendix 1). The students presented study findings to the instructor and their peers, using PowerPoint presentations. Students were encouraged to make presentations fun, interesting, and engaging. They were also responsible for leading discussion about the journal article after their presentation, with input, guidance, and questions from the instructor. Articles varied in scope and methodology, and topics were sequentially organized by complexity to align with the articles for the day's session. The Journal Club Implementation Flowchart details the process by which the instructor and students navigate each course session ( Figure 1).
The sessions were organized as follows: situate self in learning space, the day's outline, plus/delta review from the previous course session, student methods report presentation, introduction to the day's topic, 3-minute break, student journal club structured summary and presentation #1, "Who is this?," student journal club structured summary and presentation #2, and completion of the plus/delta for the course session. On 4 sessions, guest speakers with expertise provided the introduction to the day's topic, as well as served as moderators and discussants for the student journal club structured summary and presentations.
All students completed 6 methods reports or journal club structured summary and presentation peer reviews throughout the semester. They offered feedback in 3 areas: "What the presenter did well," "What the presenter could have done better," and "What additional comments do you have for the presenter?" To protect student confidentiality, there were 2 peer reviews for every presentation. The peer reviews were reviewed by the teaching assistant for appropriateness and then aggregated, blinded, and sent back to students as part of formative feedback. Students were instructed in subsequent presentations to indicate to the class 1 way they incorporated this feedback into their presentation.
Most assignments were completed independently, although a small number of students in the afternoon class completed 1 methods report presentation or journal club structured summary Friesth and Dzara 3 and presentation in teams of 2 due to a slight excess of students compared with course topics.
Course grading
Course grading was on a 1-to 100-point scale, with 1 methods report presentation (20 points), 2 journal club structured summaries and presentations (40 points), 6 peer reviews (15 points), course engagement (15 points), CITI training (5 points), and a final course reflection (5 points). After each journal club structured summary and methods report presentation, students were emailed a rubric with their grade and written formative feedback from both the instructor and their peer reviewers. The rubrics detailed whether learners followed citation and formatting instructions, clearly explained topics, and related topics to prior course content or readings. Journal club rubrics specifically indicated whether students were accurate in their interpretation of the research question, design, methods, data analysis, results, and conclusions. At the course midpoint, students were given a grade update, with their raw score as well as a scaled percentage.
Data collection
As part of routine educational evaluation and quality improvement, multiple sources of course evaluation data were collected by the instructor for educational program evaluation. These data sources included the following: 1. Anonymous, voluntary, final quantitative course quality improvement survey developed by the instructor and collected as part of routine end of the semester educational evaluation. 2. Deidentified, final 1-page course reflection assignments in which students reflected on their learning throughout the semester by drafting a 1-to 2-page reflection paper which connects course content and learning throughout the course. 3. Anonymous Plus/Delta data completed by all students after every class session to inform educational quality improvement throughout the semester. Students were asked to take 1 minute at the end of each session to answer 2 simple questions: (1) what went well, and (2) Pre-Course Instructor selects journal articles in six health sciences research areas: public health, clinical, health services, health professions education, epidemiology, patient-centered Instructor selects methods report topics: quantitative research, variables, hypothesis testing, association, statistical significance, type I and type II error, population and sample, validity, reliability, generalizability, survey research, qualitative research methods, content analysis, confidence intervals, effect size, systematic reviews, power analysis, multicollinearity, mixed methods research, research ethics Instructor assigns journal articles and dates to students Instructor prepares "self-directed structured summary" template
First Course Session
Instructor provides students with assigned journal articles and dates Instructor provides students with assigned methods reports and dates Instructor demonstrates use of "self-directed structured summary" template using a sample journal article Instructor assigns peer reviewers and explains peer review process
Each Course Session
Students upload their self-directed structured summary to the learning management system for presentation One student presents methods report presentation Two students present assigned journal articles to peers using PowerPoint Students lead and instructor supports discussion about each journal article Post Course Session Student peer reviewers submit peer reviews in learning management system Instructor reviews, collates, and anonymizes peer reviews and sends to students Instructor provides written feedback and grades to presenting students
Journal of Medical Education and Curricular Development
what could have gone better. The instructor reviewed and summarized this feedback and presented it to all students at the beginning of the next session. When possible, the instructor made small iterative changes to improve student experience. When changes could not be made, the instructor was transparent as to why.
This project was reviewed by Northeastern University Human Subjects Research Protection and exempted from further review.
Data analysis
This mixed-methods convergent educational evaluation utilizes the quantitative quality improvement survey, qualitative final course reflections, and Plus/Delta feedback as data sources.
Our purpose was to understand how the course design was experienced by students, as well understand the perceived educational impact on their understanding of and confidence with research methods. Descriptive statistics were obtained for the quality improvement survey. Plus/Delta feedback were collated and reviewed to determine key recommendations from learners. Thematic analysis following the 5 stages to qualitative research framework was utilized for the qualitative final course reflections. 24 Dedoose (QSR International Inc, Burlington, MA) was used to facilitate data management while coding. First, 2 primary coders (K.D. and M.F.) familiarized themselves with the data, each independently reviewing the first 5 reflections to create a preliminary list of codes. They compared codes and refined definitions as a team with discrepancies reconciled in person by both coders until the codebook was finalized and entered into Dedoose. Interrater reliability between the coders was established using 5 transcripts at random with a 0.75 kappa indicating good to excellent interrater reliability. Subsequently, codes were systematically applied to all reflections. After independently coding all reflections, the 2 coders discussed recurring patterns in the data and codes were combined into categories and then themes-a cluster of codes that, when combined, provided a meaningful statement about students experience in the course. The coders independently reread all coded data within each theme to ensure consistency and identify illustrative quotations. The authors then considered how the 3 data sources aligned and integrated the finding in the results to meaningfully report the impact of the course on students.
To confirm findings, a student who took the class served as a member checker by critically reviewing the manuscript prior to journal submission and providing feedback which was incorporated into the final version. The Northeastern University Institutional Review Board exempted the study.
Results
Thirty-nine students completed the quality improvement survey (97.5%) and 40 completed the final course reflection (100%). All were expected to complete the plus/delta data anonymously after each session. Table 1 presents the quality improvement survey results. These results, as well as the Plus/ Delta feedback, were integrated within the 4 themes constructed from the qualitative analysis of the final course reflection (Table 2) for results reporting.
Enabled a high degree of growth as students and scholars
Students noted a high degree of perceived growth as students and scholars. They reported gaining an appreciation for research through the course and indicated that they now knew how important it was to be a critical reader of the literature. They indicated feeling that they grew as scholars and gained academic confidence, in part through improvements in both oral and written communication. The journal club selfdirected structured summary provided an instructor-scaffolded approach that allowed the student to "chunk" the article in smaller sections, thus enabling them to understand the article as a whole. This activity required significant self-directed learning and was positively received by students. Overall, students felt enabled to read research articles and understand research methods, which they attributed in part to the methods report presentations by students during each session. This was supported by responses to multiple survey questions, which indicated that self-directed structured summaries, methods reports, and presentations were an efficacious way to teach critical appraisal. Importantly, students reported utilizing skills learned in the course in another course that term, or expectations that what they learned would be helpful in future classes or training.
Designed in thoughtful and unique format
Throughout their reflections, students offered praise for the thoughtful and unique course design, which they experienced as novel and greatly appreciated. Students found the mindfulness exercise at the beginning of each class refreshing and appreciated the opportunity reset from a busy day and refocus energy on learning. Students were highly supportive of the multiple opportunities for class discussion and felt comfortable engaging with their instructor and peers. Guest speakers were welcomed and students found them knowledgeable, inspiring, and helpful to expand their breadth of learning. The plus/delta activity at the end of each course to obtain student feedback was perceived as respectful, especially when changes were implemented during the next session. The consistent session structure was comfortable to students who also noted finding the varied course activities and variety of topics stimulating. Finally-as was also indicated in the survey-the utilization of peer teaching and learning including peer review was valued. In many cases, students incorporated this feedback into future assignments.
Initially intimidated students and was academically challenging
Students admitted to entering the course with a high degree of apprehension and anxiety about the topic and did not feel prepared by prior courses. They expected to be bored and uninterested. They noted feeling overwhelmed by the syllabus and apprehensive about the multiple required presentations. Once the course was underway, they felt academically challenged by the course and assignments, and felt the level of work required for the course was appropriate. Some disliked the methods report presentations and felt it was of lower educational value than other course activities.
Prioritized and enabled psychological safety
Students reported not being "stressed out" as a function of the course through the safe, welcoming, and comfortable learning environment created and sustained throughout the semester. They felt they could learn and grow and that the instructor and, subsequently, their peers were invested in their academic growth. They were comfortable engaging in discussion and noted doing so more than in other courses. Plus/Delta feedback was consistently received and was transparent-for example, offering instances when articles were too complex to facilitate learning, or times when the instructor may have been more effective in explaining key concepts. When possible, this immediate feedback informed iterative course changes, such as implementation of a 3 minute course break for restroom use and reduction in number of course readings. Overall, feedback was overwhelmingly positive, with students indicating that the instructor was high-energy and invested in their growth, and that their time and input as students was respected. These findings were supported by survey results, in which nearly all students reported the course being a positive learning experience worth their time and effort. Importantlyand further evidence of the psychological safety enabled through the course-students noted that future improvements could include a wider variety of in-class activities, inclusion of a group project, more integration between the methods report and journal club structured summaries, and additional engagement through discussion and the limiting of student laptop use.
When considered in the aggregate, the 3 data sources converge and highlight the perceived positive impact of the course on learners, who found it challenging yet noted that the class was worth their time and effort. Moreover, they ultimately no Yes I would recommend this course to a friend in the health sciences major 0 (0%) 39 (100%) note: Thirty-nine students completed the quality improvement survey. Statements were rated on a 5-point Likert-type scale ranging from strongly disagree (1) to strongly agree (5).
Appreciation for Research and Desire to Be a Critical Readers
"I also appreciated research much more as a field of study and as a professional concentration." (Reflection 2) "I now feel comfortable with reading these papers, and thus being critical about the data, methodologies, and analysis that the authors use." (Reflection 18)
Perceived Growth as Learners and Scholars
"This class challenged me not only academically but also personally. Throughout this semester I was able to learn, grow as a scholar and as a person." (Reflection 1) "One of my greatest lessons from the class was learning how to improve my writing and oral presentation skills through reading papers and presenting." (Reflection 1)
Journal Club and Methods Reports Approach Enabled Understanding of Research Methods
"Journal Club was most effective in helping me become a better critical reader of research articles and identify the research question, research methods, and study design." (Reflection 7) "The methods reports also helped me learn about health care research in a unique way." (Reflection 15)
Application of Knowledge in Current and Future Coursework
"I didn't realize how much I was learning in this class, until one day, when I was reading through research articles for a project in my genetics class. As I was going through different articles, I thought, 'wait, I am understanding what these papers are saying. This feels much easier than any research I have done before.' I think this was a direct result of the knowledge I was gaining through this class." (Reflection 5) THEME 2: DESIgnED In THOUgHTFUL AnD UnIQUE FORMAT
Learners Appreciated the Unique Design which Enabled Learning
"I firmly believe that this class was one of the most insightful and relevant classes that I'll attend at northeastern." (Reflection 11) "Having the plus/deltas at the end of every class made me feel like a more active participant in my learning and someone who's opinions were valued. I could see the class improving each time I came, instead of holding on to issues or concerns until the end of the semester." (Reflection 3)
A Consistent Course Format with Some Variety Engaged Learners
"Having a varied format was a great way to keep each class interesting. Having a similar structure each time made each class predictable enough to be comfortable, but different enough to keep my attention. It became familiar to have a methods report, a methods lecture, a journal club presentation, a "who is this," another journal club, then our plus-deltas." (Reflection 2) Peer Teaching, Review, and Feedback was Appreciated, Valued, and Implemented "Having to present in front of my classmates and receive critiques on my presentation of the journals and methodological topic definitely enhanced my public speaking skills and allowing me to speak with confidence when I am discussing research findings in studies." (Reflection 30) "Providing feedback was not only helpful to the person receiving the feedback, it also allowed me to able to learn from other presenters to improve my own communication skills." (Reflection 7)
THEME 3: InITIALLY InTIMIDATED STUDEnTS AnD wAS ACADEMICALLY CHALLEngIng
The Course Was Academically Challenging and Initially Intimidating "To be completely honest, after the first day of this class, I was dreading the rest of the semester. First, I thought I already knew that research was not something I was interested in pursuing. Second, after hearing that most of our final grade would be based on these 'journal club' and 'methods report' presentations, I was terrified." (Reflection 5) "I found this course to be challenging both in the content and the assignments" (Reflection 36) THEME 4: PRIORITIzED AnD EnABLED PSYCHOLOgICAL SAFETY
The Learning Environment was Comfortable and Supportive
"As the semester went on, I began to realize that this class fostered a supportive learning environment." (Reflection 35) "I could tell everyone wanted to be there each class, and I felt comfortable approaching classmates with questions outside of class or for further discussion of topics we addressed during class time." (Reflection 3) 7 developed great respect for the instructor and the course design, and would recommend it to a friend in the same major. Perhaps most importantly, they both reported and were proud of their own subsequent resultant growth as scholars and researchers.
Discussion
We describe the development, implementation, and evaluation of a undergraduate health care research course utilizing a journal club format which Generation Y students indicated allowed them to better understand research articles and improve as scholars. Psychological safety was reinforced consistently and students expressed comfort in and out of the classroom that led to their overall success in the course. Students also felt appreciated throughout the course, which in turn encouraged them to be more involved and therefore they were able to reach a higher level of learning. Students also greatly appreciated that the instructor maintained a consistent structure of the course while still offering varied activities and covering diverse topics in each session. Importantly, students noted that improvement was possible and gave multiple recommendations for how that could be achieved. Importantly, in the United States, the Accreditation Council for Graduate Medical Education mandates that programs support advancement in resident knowledge of basic scientific inquiry principles, including how research is designed, conducted, assessed, explained to patients, and applied to patient care. 25 Those trainees who have a strong undergraduate research experience may be more likely to readily engage in research and quality improvement projects as trainees, and may be more equipped to encourage critical thinking and evidence-based medicine among their peers.
Our study has multiple limitations. First, this course was implemented in just 2 sections at 1 university and thus may not translate to other settings. Moreover, data from 2 course sections were combined for more robust data analysis and differences in student composition may have influenced class culture. The survey does not have existing validity evidence and was created by the instructor. We also did not design our study as research but instead report the evaluative results of an educational intervention. Finally, students were not asked how their learning experience compared to other courses at their institution, which offered no counterfactual or benchmark from which to compare the course evaluation.
Conclusion
Teaching styles that focus heavily on didactic teaching are misaligned with the teaching and learning desires for Generation Y students. The student-led "journal club" course structure scaffolded with a self-directed structured summary template allowed students to gain mastery over the challenging task of critically appraising health care research articles in a way which was meaningful and impactful to them. This educational evaluation demonstrates evidence that the course structure was wellreceived by students and resulted in perceived growth as students and scholars. We attribute our success in part due to the course's high degree of psychological safety, which was noted, appreciated, and valued by students. | 2020-07-23T09:09:33.627Z | 2020-07-01T00:00:00.000 | {
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33448128 | pes2o/s2orc | v3-fos-license | Electric Dipolar Kondo Effect Emerging from Vibrating Magnetic Ion
When a magnetic ion vibrates in a metal, it inevitably introduces a new channel of hybridization with conduction electrons and in general, the vibrating ion induces electric dipole moment. In such a situation, we find that magnetic and non-magnetic Kondo effects alternatively occur due to the screening of spin moment and electric dipole moment of vibrating ion. In particular, electric dipolar two-channel Kondo effect is found to occur for weak Coulomb interaction. We also show that magnetically robust heavy-electron state appears near the fixed point of electric dipolar two-channel Kondo effect. We believe that the {\it vibrating} magnetic ion opens a new door in the Kondo physics.
It has been widely recognized that Kondo phenomena generally appear when localized entity with internal degrees of freedom is coupled with conduction electrons. Concerning the original problem of resistance minimum phenomenon in metals with magnetic impurities, Kondo has actually shown it by quantum-mechanical calculations for scattering amplitude of electrons due to magnetic impurities [1]. Then, it has been revealed that the singlet state is formed from local magnetic moment due to the coupling with conduction electrons [2]. After the understanding of the Kondo effect in the dilute magnetic impurity system, interests of researchers have moved to the impurity with complex degrees of freedom.
One research direction has been found in the explicit consideration of orbital degree of freedom of localized electron. Coqblin and Schrieffer have derived exchange interactions from the multiorbital Anderson model [3]. Then, the concept of multi-channel Kondo effect has been developed on the basis of such exchange interactions [4], as a potential source of non-Fermi liquid phenomena. Such non-Fermi liquid properties have been pointed out also in a two-impurity Kondo system [5,6]. Concerning the reality of two-channel Kondo effect, Cox has pointed out the existence of two screening channels in the case of quadrupole degree of freedom in a cubic uranium compound with non-Kramers doublet ground state [7].
Another possibility is non-magnetic Kondo effect with phonon origin. First Kondo has considered Kondo-like behavior in a two-level system [8]. The two-level Kondo system has been proven to exhibit the same behavior as the magnetic Kondo effect [9]. Yu and Anderson have discussed the phononic Kondo effect from a different viewpoint [10]. They have pointed out that the scattering process between spinless s-wave conduction electron to p-wave one is produced by ion displacement.
Recently, the Kondo effect with phonon origin has attracted renewed attention due to active experimental investigations on cage structure materials, in which a guest ion is vibrating in a cage composed of relatively light atoms. We believe that the vibration of magnetic ion in the cage provides a new ingredient in the Kondo physics, since dynamical aspects of magnetic impurity have been considered unsatisfactorily in the Kondo problem. In fact, quite recently, the two-channel Kondo effect has been confirmed in the model for vibrating magnetic impurity [11][12][13][14]. Also for the promotion of our understanding on magnetically robust heavy-electron phenomenon observed in cage compound [15], we further develop the Kondo physics of vibrating magnetic impurity.
In this Letter, we analyze a two-channel conduction electron system hybridized with vibrating magnetic ion by using a numerical renormalization group technique. We confirm magnetic and non-magnetic Kondo effects originating from the screening of spin and electric dipolar moments, respectively, by evaluating entropy and susceptibilities for spin and electric dipole moments. Near the fixed point for electric dipolar two-channel Kondo effect, we find magnetically robust heavy-electron state from the direct evaluation of the Sommerfeld constant.
Let us consider a two-channel conduction electron system hybridized with vibrating magnetic impurity [12][13][14]. In the unit ofh=k B =1, the Hamiltonian is given by where ε k denotes conduction electron dispersion, c kℓσ indicates the annihilation operator for conduction electron with momentum k, angular momentum ℓ, and spin σ, f σ is the annihilation operator for localized electron with spin σ, n σ =f † σ f σ , U is the Coulomb interaction between localized electrons, E f denotes the local f -level energy, V 0 is the hybridization between s-channel conduction and localized f electrons, g is the electron-vibration coupling, x denotes the ion displacement, p indicates the corresponding canonical momentum, and ω is the vibration frequency. Note that we set the reduced mass of vibra- tion as unity. In order to consider the symmetric case, we set E f =−U/2 throughout this paper.
In this paper, we analyze the model with the use of a numerical renormalization group (NRG) method [16]. We introduce a cut-off Λ for the logarithmic discretization of the conduction band. Due to the limitation of computer resources, we keep N low-energy states. In this paper, we set Λ=5 and N =5000∼10000. Note that the temperature T is defined as T =Λ −(i−1)/2 in the NRG calculation, where i is the number of the renormalization step. The phonon basis is truncated at a finite number N ph , which is set as N ph =300 in this paper.
It is convenient to introduce phonon operators a and a † through the relation of x=(a + a † )/ √ 2ω. Then, we define the magnitude of phonon-assisted hybridization as The energy unit is the half of the conduction electron band D, which is set as unity. As for parameters, we set V 0 =ω=0.2 and we change the values of U and V 1 .
First let us visualize the situation described by the present model. In Fig. 1, the two horizontal lines symbolically denote s and p channels which are hybridized with magnetic impurity. As shown in H, s-channel electrons are hybridized with localized electron in a standard manner, while the hybridization process between p-channel and localized electrons is assisted by phonons. Note that parities of s-and p-channel electrons are different, as easily understood from the values of angular momenta. Namely, s-and p-channel electrons possess even and odd parities, respectively. Since the parity for ion displacement x is odd, it is natural that the phononassisted hybridization occurs only for p channel.
When magnetic ion stops at an origin, we consider only the screening of impurity spin moment by s-channel electrons, leading to the conventional Kondo effect. However, when the ion is vibrating as shown in Fig. 1, there occurs another screening process due to p-channel electrons. In such a situation, in addition to the screening of spin moment, the electric dipole moment in proportion to the displacement x should be also screened by conduction electrons, leading to non-magnetic Kondo effect. Intuitively, we understand that the main screening channel is converted between s and p due to the balance between V 0 and V 1 . In fact, the phases with different quantum numbers in relation with parity have been found to be converted between the regions of large and small V 1 . Then, two-channel Kondo effect has been confirmed to occur just at the boundary between those two phases [11][12][13]. However, another conversion between magnetic and non-magnetic Kondo effects controlled by the balance between U and V 1 has not been clarified yet. Then, in this paper, we unveil such a new point in relation with magnetically robust heavy electron state. Now we explain the phase diagram in Fig. 2(a). As mentioned above, the solid curve indicates the boundary between two phases with different quantum numbers, which are obtained by the s-and p-channel Kondo screening, respectively. On the boundary curve between two phases, the two-channel Kondo effect is realized. Since no phase conversion occurs on the line of U =0, the boundary curve asymptotically approaches the line of U =0. Note that low-energy spectra of three fixed points of s-channel, p-channel, and two-channel Kondo states have been revealed in Refs. [12] and [13]. New results of this paper are color gradation and other broken and dotted curves. Their meanings will be discussed later.
Next we discuss the NRG results of entropy. In Fig. 2(b), we show the change of entropy along the lines of U =2, V 1 =0.2, and U =0.05. On the line of U =2, for V 1 =0.14 and 0.2, we find the plateaus of log 2, which are eventually released at low enough temperatures. At (V 1 , U )=(0.175, 2), we observe the entropy of 0.5 log 2 at low temperatures, which is the signal of two-channel Kondo effect. When we decrease the value of U from U =2 on the line of V 1 =0.2, we observe the increase of the Kondo temperature T K , which is characterized by the release of entropy log 2. This is quite natural from the viewpoint of the conventional magnetic Kondo effect. Thus, we deduce that the Kondo effect on the line of U =2 as well as in the region of U > ∼ 1 on the line of V 1 =0.2 is originating from the screening of impurity spin moment.
However, for U < ∼ 1, a plateau of log 2 again appears. The temperature region of log 2 is wider for smaller U and T K is decreased with the decrease of U . Such behavior is contradictory to the conventional magnetic Kondo effect. Furthermore, at (V 1 , U )=(0.17384, 0.05), we again observe a clear plateau of entropy of 0.5 log 2, but it is difficult to understand the origin of this two-channel Kondo behavior only from the result of entropy.
In order to clarify what quantity is screened, we evaluate susceptibilities for magnetic and electric dipole moments, which are, respectively, defined by Here · · · denotes the operation to take thermal average, M (τ )=e Hτ M e −Hτ , M =g s µ B (n ↑ −n ↓ )/2, and P =Ze(a+ a † )/ √ 2ω, where g s is the electron g-factor which is set as g s =2, µ B is the Bohr magneton, Z is valence number of ion, and e is electric charge. In the actual calculations, we normalize them as χ M /µ 2 B and χ P /(Z 2 e 2 /2ω). In Fig. 3(a), we show T χ M for the same values of U and V 1 in Fig. 2(b). As we have deduced above, on the line of U =2, we observe the decrease of T χ M around T K . Note that the curves of T χ M for V 1 =0.14 and V 1 =0.2 at U =2 are quite similar at low temperatures, when T is rescaled by T K . On the other hand, the curve of T χ M for (V 1 , U )=(0.175, 2) is apparently different from those for V 1 =0.14 and V 1 =0.2. It is due to the non-Fermi liquid behavior in the two-channel Kondo effect, leading to χ M ∼− log T . This logarithmic correction in χ M is clearly observed. Along the lines of V 1 =0.2 and U =0.05, T χ M decreases at relatively high temperature, but the release of the entropy does not seem to correspond to the temperature at which T χ M is decreased. Now we turn our attention to the results for T χ P in Fig. 3(b). The entropy release for (V 1 , U )=(0.2, 0.05) corresponds to the decrease of T χ P , indicating the occurrence of the Kondo effect concerning electric dipole moment. Note that χ P is related to phonon Green's function. In the strong electron-phonon coupling region, the center of oscillation is shifted either right or left, leading to χ P ∝q 2 /T , where q= (a + a † ) 2 . Thus, for small U and large V 1 region, we expect that T χ P becomes constant at high temperatures, as found in Fig. 3(b).
When T is decreased, T χ P is decreased from the constant value due to the Kondo screening of electric dipole moment and it eventually goes to zero at T =0. This can be called the parity Kondo effect, since near degeneracy with different phonon parities characterizes the electric dipole, which is coupled with conduction electron parity, leading to the non-degenerate ground state with fixed total parity. Note that the total parity is specified by 0 or 1, depending on U and V 1 . At low enough temperatures, we find T χ P =2T /ω 1 , whereω 1 is renormalized phonon energy smaller than ω. From the local Fermiliquid theory, we find T K ∝ω 1 , but the proportional coefficient is suppressed by the polaron effect in comparison with the conventional magnetic Kondo effect. Then, T χ P decreases rapidly around T K in sharp contrast to T χ M around T K . The shape of T χ P at (V 1 , U )=(0.2, 1.0) around T K is quite similar to that for (0.2, 0.05) [17], when T is rescaled by T K . For the case of (0.2, 1.0), corresponding to the competing region of magnetic and electric dipolar Kondo effects, we do not find any significant structure in the entropy and T χ M , but in the decrease of T χ P from the shoulder, the enhanced signal can be observed due to the polaron effect in the phonon matrix element of χ P . We remark that the two-channel Kondo effect also occurs due to the screening of electric dipole moment [18]. In fact, at (V 1 , U )=(0.17384, 0.05), we find a plateau of entropy 0.5 log 2 and the significant decrease of T χ P around the corresponding temperature. Note that the shape of T χ P at (0.17384, 0.05) is different from those for (0.2, 0.05) and (0.2, 1.0). We observe the smooth change from T χ P =constant to χ P =constant in this case. Since the electric dipole moment is not perfectly screened, vibration still remains and thus, we intuitively obtain χ P =2/ω 2 in the two-channel electric dipolar Kondo regime, whereω 2 is another renormalized phonon energy, which is different fromω 1 . It is one of future tasks to explain the difference betweenω 1 andω 2 by overcoming the difficulty to estimateω 1 with high precision in the NRG calculation.
Here let us go back to Fig. 1(a). We have found that the electric dipole moment is screened in the region of large V 1 and small U . In order to visualize the change of the screened moment, we depict q as the color gradation in Fig. 1(a). For large V 1 and small U , we find the red region with large q, in which electric dipolar Kondo effect occurs. On the other hand, there occurs magnetic Kondo effect in the blue region of small V 1 and large U . As a guide of the boundary between magnetic and non-magnetic Kondo regions, we plot inflection points of d 2 q/dU 2 =0 (dotted) and d 2 q/dV 2 1 =0 (broken) in Fig. 1(a). For large V 1 , two curves run in the green area between blue and red regions. Another broken curve appears on the two-channel Kondo line for U < ∼ 0.6, except for U =0, suggesting the electric dipolar two-channel Kondo region. Note, however, that the inflection points seem to lose the meaning of boundary in the s-channel Kondo region, since renormalized Fermi chain is realized in the region of small U and small V 1 [12,13]. In the small area of yellow and orange of the s-channel Kondo region, there also occurs non-magnetic Kondo effect, which is interpreted as the Yu-Anderson Kondo effect [10]. Now let us discuss the magnetically robust heavy electron state by the Sommerfeld constant γ. For the purpose, we add the Zeeman term H Z =g s µ B H(n ↑ − n ↓ )/2, where H is an applied magnetic field, to the model (1). Then, we evaluate γ at T =Λ −14 , the lowest temperature at which we can arrive in the present NRG calculations. In Fig. 4(a), we show the results for γ in the unit of mJ/mol · K 2 with D=1 eV. We find that γ shows divergent behavior around the two-channel Kondo fixed points, shown by the solid curve in Fig. 1(a). On the line of U =0, we do not find the phase conversion, but for V 1 > ∼ 0.17, the two-channel Kondo line exists in the extreme vicinity of U =0. Thus, γ becomes very large for U =0 and V 1 > ∼ 0.17. In the vicinity of the two-channel Kondo line, we find the enhancement of γ due to the non-Fermi liquid properties, irrespective of U . For the parameters away from the two-channel Kondo fixed points for U >0.05, γ in the region of the magnetic Kondo effect is relatively large in comparison with that of the nonmagnetic Kondo effect of electric dipole moment.
In Fig. 4(b), we show the ratio of γ's at H=0 T and 30 T. If this ratio is near the unity, we judge that γ is magnetically robust. As we easily imagine, in the Kondo effect concerning the electric dipole moment, γ does not depend sensitively on the magnetic field. As for criteria of the magnetically robust heavy electron state, we consider the conditions of γ>1000 and γ(30T)/γ(0T)>0.9, shown by horizontal broken lines in Figs. 4(a) and (b). After the NRG calculations, we find that the magnetically robust heavy electron state appears in the region of V 1 > ∼ 0.17 and U < ∼ 0.1, except for the narrow region near the two-channel Kondo line. Note that the ratio is strongly suppressed due to the divergent behavior of γ(0T). That region is included in the non-magnetic Kondo effect region with orange and red in Fig. 1(a). In comparison with the mechanism of magnetically robust large γ on the basis of the charge Kondo effect [19], it seems to be easier to obtain large γ in the present scenario based on the nonmagnetic two-channel Kondo effect.
In summary, we have analyzed the two-channel conduction electron model with vibrating magnetic ion. We have found two types of Kondo effects due to the alternative screening of magnetic and electric dipole moments. Near but not exactly on the two-channel Kondo line with electric dipolar origin, we have found that γ is magnetically robust. Non-magnetic electric dipolar Kondo behavior is expected to be observed in cage-structure materials. In particular, magnetically robust non-Fermi liquid behavior is an interesting possibility. This work was supported by KAKENHI (20102008). The computation in this work has been done using the facilities of the Supercomputer Center of Institute for Solid State Physics, University of Tokyo. | 2012-03-21T09:47:05.000Z | 2012-03-21T00:00:00.000 | {
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233275025 | pes2o/s2orc | v3-fos-license | The rise of electronic commerce in healthcare: The need to ensure consumer safety
Houda Attjioui*1, Amine Cheikh2, Amina Tebaa3, MohamedWadie Zerhouni4, Zineb Aliat1, Ha id Mefetah5, Mustapha Bouatia6 1Faculty of Medicine and Pharmacy in Rabat, Mohammed V University in Rabat, 10100, Rabat, Morocco 2Department of Pharmacy, Abulcasis University of Health Sciences, Cheikh Zaid Hospital, 10100, Rabat, Morocco 3Department of Pharmacovigilance, Moroccan anti-poison and Pharmacovigilance Center, Ministry of Health, Rabat, Morocco 4Department of Drugs and pharmacy (DMP), Ministry of Health, 10100, Rabat, Morocco 5Department of Pharmacy, Pediatrics hospital, 10100, Rabat, Morocco 6Faculty of Medicine and Pharmacy in Rabat, Team of Formulation and Quality Control of Health Products, Laboratory of Analytical Chemistry, Mohammed V University in Rabat, 10100, Rabat, Morocco
Heath Product, e-commerce, internet, illegal sales, counterfeiting, patient safety A The growth of e-commerce is revolutionizing access to health products, and offers of these products on the Internet are often attractive from the customer's point of view. However, it contributes signi icantly to the expansion of international trade in falsi ied medicines. The objective is to take stock of the signi icant growth in the online sale of health products with the health challenges encountered. We conducted a survey on the purchase of the most popular online health products in Morocco, and then examined the type of health products offered, the price of the most common drugs compared to conventional prices.49 websites and social network forums were identi ied, 37% of which had a speci ic address and 63% of which had a virtual interface. The sales strategies identi ied are numerous, the main ones being the availability of medical specialties not available in the country, the order to pay less and save time. The most common purchases concern so-called "comfort" medicines.
INTRODUCTION
The growth of e-commerce in the world is unstoppable. With double and even triple-digit growth since its introduction in the 1990s, it has become easy to ind everything online that the customer wants to buy immediately Levaggi et al. (2009). The growth of the internet and e-commerce has also taken on signi icant importance in the ield of health, from the simple dissemination of information to a real opportunity to acquire health products directly ).
Many websites, blogs, and forums are emerging on the web to highlight the bene its of e-Health services, or to promote exchanges between Internet users to ind the "best" addresses or to compare prices. Besides, the behaviour of many Internet users has evolved towards the sale of various pharmaceutical products over the internet, which are often considered suspicious, most often associated with counterfeits (Lavorgna, 2015;Liang and MacKey, 2009).
Many pharmaceutical products have gradually been offered, and their online purchase can offer signi icant bene its that are often attractive from the customer's point of view, including, as recognized by the Us Food and Drug Administration (FDA), a "private and convenient way to obtain the necessary drugs, sometimes at affordable prices". However, risks to consumers are signi icantly increased when obtaining medical products from unlicensed and unregulated sources (Fittler et al., 2013).
The World Health Organization (WHO) is facing this problem in the context of counterfeit medicines, de ined as "a global public health crisis", stating that medicines purchased over the internet from sites hiding their physical address are counterfeit in more than 50% of cases (of the World Health Organization, 2010). The WHO, for its part, has announced some alarming igures: Globally, one out of every two medicines sold on the internet is, in fact, a counterfeit, causing more than 700,000 deaths each year. A criminal but extremely lucrative business, with a turnover estimated at more than 75 MM dollars (Organization, 2017).
Although the drug supply chain is highly regulated in most countries of the world, Morocco is not immune to this global phenomenon of illicit drug sales. Article 30 of the Law No. 17-04 on the code of the drug and pharmacy, certainly establishes the obligation to sell medicines in pharmacies, but the non-conventional circuit remains important and presents serious dangers for the health of citizens (of Morocco, 2006).
Today, there is a remarkably wide range of websites on the web that distribute, almost always illegally, different families of medicines and health products. Many people continue to buy medicines informally, unaware that these medicines can be dangerous because their composition is not subject to any marketing authorization, quality control, expiry date, or controls on transport and storage conditions. Illegal sales of health products encompass several aspects, including the sale of real medicines by unauthorized actors, the sale of counterfeit products, the sale on the market of products whose storage and transport do not comply with regulations, prohibited or MA-free medicines (Jena et al., 2011).
From these elements, other issues such as expired and relabelled drugs, contraband drugs, over-thecounter drugs purchased outside pharmacies...".
Africa is probably the continent that pays the highest price for the traf icking of counterfeit medicines, The scourge of counterfeiting mainly affects some regions of Africa, Asia, and Latin America, where access to medicines is limited (Organization, 2006). Distribution channels are much less controlled, allowing illegal networks to enter the out in two phases: the irst phase consisted of collecting and analyzing the existing market more ef iciently.
Thus, the objective of the study was to take stock of the signi icant growth in the online sale of health products with the health challenges encountered and to propose actions and perspectives adapted to public health challenges.
MATERIALS AND METHODS
The analysis of this study was carried publicly accessible data, from a selection of the most frequently cited websites, forums, blogs in Morocco and collecting messages on social networks that offer different health services and offers, focusing in particular on the characteristics of promoters and their promotional strategies, asking whether they declared their detailed physical locations with an apparent geographical reference or only had a virtual interface.
Data collection included all websites speci ic to the sale of pharmaceutical and medical products, the set of websites allowing the publication of ads of different categories of service: sale, rental, employment (example: Avito, Jumi), and some Social networks: Facebook, Twitter, and Instagram.
The search was conducted using some pre-selected keywords that were a combination of medical products and terms that mentioned the marketing and presumed sale of drugs and health products.
In a second step, we examined the type of health products proposed, the price of the most frequently present drugs compared to conventional prices and the origin of the products proposed.
The data collected allowed us to identify the problem and study the courses of action to be considered to secure the purchase of drugs.
RESULTS AND DISCUSSION
According to the report of the Moroccan anti-poison and Pharmacovigilance Center, drugs are the leading cause of intoxication in Morocco. Nearly 4108 drug poisonings, which resulted in 4 deaths, were recorded in 2017, 65% of these poisonings are accidental (31% are due to fake drugs), compared to 35% of suicide attempts. The people most affected were adults (47.7%, compared to 52.19% overall) and baby walkers (22.9%), according to the report of the Moroccan anti-poison and Pharmacovigilance Center.
Characteristics of promoters and contact details
We identi ied 49 websites, forums, and blogs on social networks that meet the inclusion criteria, including 37 % with a speci ic address and 63% with a virtual interface. Only 39% of the promoters provided full details of their physical location, with 63% reporting that they were in Morocco, 31% in Europe, and 6% in Turkey.
56% of promoters declaring their physical location offer home deliveries to the applicant regardless of their country or city of residence. In other forums, such as FACEBOOK, advertisers offer their products by asking interested parties to contact them either by the phone number they post or to contact them privately for more details.
Marketing strategies
The analysis of sales promotion strategies revealed that the most common ones were the safe use of personal data and secure payment, con identiality, high product quality with affordable prices, short delivery times and delivery facilities, which offers comfort to the customer, as well as a suggestion for a bulk purchasing to pay less for some advertisers. The strategies identi ied are shown in Table 1.
We have also noticed that these offers are accompanied by a large number of responses in different forms: testimonial opinions or comments from various sources.
It is also noted that sales promotion strategies differ between a virtual interface and a physical pharmacy and that the strategy of buying cheaper remains the most common point for both promoters regardless of its location. This is represented in Table 2.
Products offered Online
The most common purchases concern in particular so-called "comfort" medicines indeed, products intended to treat sexual problems (23%), in particular, erectile dysfunction (Sildena il), female hormones (5%), slimming products for weight loss (11%), food supplements or vitamins (8%), products for the relief of joint and muscle pain (4%) and cosmetic products (35%) are the products most concerned. The classi ication of products offered online is represented in Table 3.
Among the drugs that circulate on social networks are speci ic high-risk drugs such as Insulins, Tacrolimus monohydrate, Enoxaparin, Norditropin, Minoxidil, Cyproheptadine with vitamins, Posal ilin The drugs found are given in Table 4.
These drugs are found on social networks where the majority sold only one or two drugs at most. These are drugs sold through virtual interfaces without the required medical prescriptions.
These announcements do not include any information on the quality and compliance with the storage conditions of these sensitive products.
Prices
The price of speci ic drugs offered for some Internet users was lower than the price of the legal circuit, with a price difference ranging from 5 to 400 Moroccan Dirhams (MAD) ( Table 5), which attracts the customer more towards buying these products via the internet. For others, prices have not been speci ied, and ask interested parties to contact them for more details.
Origin of the products offered
The majority of imported health products come from producing countries (85%) India, China, Turkey, and Russia and then resell them at a lower cost in other countries. One example of promotion found "Our website provides quality generic medicines shipped directly from India. Each of the products available on our website is manufactured with the best raw materials and with the greatest care in world-class pharmaceutical manufacturing plants in India".
Discussion
Counterfeit medicines claim many victims in Morocco, as drug poisoning is second only to food poisoning. However, the Antipoison and pharmacovigilance centre considers that the igures recorded remain below reality insofar as the adverse events and deaths that occur at home escape the centre's reporting system, this underreporting of the patient to his doctor or pharmacist can be considered in a context of shame linked to this unauthorized purchase.
These drug poisonings are related mainly to the consumption of counterfeit drugs, counterfeit products, products of poor quality whose manufacture, storage, and transport do not comply with standards or products that have expired and been relabelled. It is therefore not surprising that the internet has become one of the primary means of marketing these counterfeit medicines. Indeed, one out of every two drugs sold on a website that does not declare its physical address is a fake (Desai, 2016). However, the declaration of geographical location is an essential feature concerning transparency.
Orizio found that online pharmacies requesting a prescription were signi icantly more likely to report their geographic location than online sales sites that did not, which were most often just virtual interfaces. These results are consistent with the results found in our study, where most of the promoters who did not report their location were mostly virtual interfaces.
The irst reason for buying medicines online is the accessibility of medical specialities not available in the country. Then there is the concern to pay less and save time or to be able to buy in complete discretion. The literature review also shows that different sales strategies are adopted by Internet users, depending on the products.
Levaggi and Orizio disclosed the promotional strategies used more frequently by online sales sites to sell their products. Identi ied arguments regarding privacy, quality of services and drugs, price offers assurance that buying drugs online is legal, and the suggestion that customers can get a drug while avoiding a visit to the doctor (Levaggi et al., 2009;Orizio et al., 2009). In particular, privacy concerns related to the use of personal data and discreet packaging; quality of service statements related to short delivery times, online tracking of order status and, indirectly, the posting of testimonials from people who have already purchased online; and price offers mentioned as an incentive to buy in bulk. Armstrong (Armstrong et al., 1999) reported that one-third of their sample highlighted the bene its of online ordering, including con identiality, ease of ordering, and cost reduction.
Other indings reveal contradictions. Products were offered at more advantageous prices if the customer had a prescription, while for other products, it was the absence of a prescription that was synonymous with a reduction. These different strategies seem to be designed to promote pro its rather than compliance with legislation.
It seems today that online sales have become more and more complex as they offer almost every type of product. In contrast, a few years ago, they tended to sell mainly lifestyle products, such as sildena il and food supplements.
We are therefore witnessing a diversi ication between the offer of medical products by the different Internet users-this with prices signi icantly lower than those charged in traditional pharmacies. The packaging is deceiving from the outside and inspires con idence in uninformed consumers. The message accompanying the proposed offer and the price are undoubtedly irresistible.
Besides, shipping and handling fees and online consultation fees can reduce total savings for consumers (Müller et al., 2009) who complain about the high prices of these products in pharmacies, the unavailability of the product, and the requirement to submit a prescription. This market for health products is lourishing and is attracting more and more young people precisely.
It should be noted that, to date, the sale of medicines (of Morocco, 2006) and prohibits any sale outside conventional channels, which remains essential and presents severe dangers to the health of citizens.
Medicines purchased on the internet even when offered under a known name already marketed are not always those for which a marketing authorization has been granted. Distribution channels used for the sale of medicines on the internet are not, as a general rule, part of the pharmaceutical chain regularly controlled by the health authorities.
According to a WCO survey, in 2011, there are 106 producer countries from all the continents, including Brazil, Argentina, Indonesia, Russia, Ukraine, Egypt or the Philippines, and 146 destination countries. However, according to recent research, Asia, China, and India in particular, are the principal manufacturers of counterfeit drugs (50% to 70%, according to estimates) (Organization, 2017). Both countries developed an important pharmaceutical industry at the time of the legal recognition of generic drugs in the 1970s. For example, India now has more than 20,000 drug producers, most of them small and specialized in generics, and about 800,000 distributors (Swaminath, 2008). In such a large sector, attempts at regulation and control are not very effective against the parallel industry.
Several recent reports of the use of medicinal products outside their marketing authorization and serious adverse reactions related to the use of falsi ied medicinal products purchased via the internet have been recorded.
Since 2013, WHO has received more than 1500 reports of substandard or falsi ied products, most of these reports (42%) come from the WHO Africa region. Falsi ied or fake medicines may contain insuf icient quantities of active ingredients to be effective. In other cases, they do not contain any active ingredients, or even impurities or toxic substances. While inactive drugs do not affect the diseases they are supposed to treat, those containing active ingredients are still too often harmful and not only to patients (Organization, 2017).
A literature review by Gorizia (Orizio et al., 2011) revealed a dozen articles published with about sixty cases concerning the purchase of drugs on the internet, including about twenty related to the consumption of products to lose weight. Neuberg et al. (Neuberg et al., 2009) reported one case of lifethreatening thyroid hormone abuse in a 56-year-old female patient encouraged and facilitated by unconventional health advice. Also, in the United Kingdom, an acute coronary syndrome was diagnosed in a 41years old male who had taken Viagra for erectile dysfunction (Solomon et al., 2002).
Pharmacovigilance, in this case, cannot be exercised a priori on these health products since their marketing is not reported to the health authorities. Therefore, risks are often only identi ied as a posteriori in the context of market surveillance or when an alert has been transmitted to the health authorities, and the link has been established with the product (Badrane et al., 2018).
The purchase of health products, particularly medicines on the internet, should be made more secure. Different actors are likely to intervene to combat the illicit sale of these products, each playing their role.
These indings highlight public health issues and prospects for better regulating the purchase of health products, particularly medicines on the internet.
On the one hand, it is important to avoid customers from going to illegal websites that offer all kinds of pharmaceutical products. On the other hand, it is necessary to create a simple regulatory framework to de ine better the rules governing the purchase of medicines via the internet to keep the medicine in a circuit authorized and delivered by a regulated professional.
For several months, the Ministry of Health has been working on a draft text on the legal sale of medicines and strengthening the ight against counterfeit medicines. A reinforced action plan with the various actors in the ight against counterfeit medicines is also underway.
Indeed, strict regulation of medicines and its enforcement by national drug regulatory authorities would contribute signi icantly to the prevention and detection of counterfeiting.
At the national level, some perspectives are possible Development of operational cooperation between the drug industry, pharmacists, and the customs intelligence department, Gendarmerie, Judicial Police to combat the illegal sale of drugs.
Development of cooperation with the public authorities and the national drug control laboratory with technical data sheets to assist in the identi ication and control of the main suspect products ; Setting up an active monitoring unit on the internet and investigation service.
Carrying out campaigns to warn against the risks associated with the purchase of medicines from illegal sources, via the internet.
Implementation of surveillance and sanction measures to identify and prohibit illegal sales and counterfeiting and other falsi ications of medicines, in collaboration with the judiciary, police, gendarmerie, and customs services, Reinforcement of the capacity to detect orders from the Internet and control postal parcels.
Besides, awareness-raising campaigns aimed at the general public by the Ministry of Health could be deployed in other regions. Websites, through an information page, can raise awareness among the general public about the risks associated with the purchase of medicines online by encouraging them to favour of icial distribution channels, to double their vigilance in the face of an abnormally low price, the presence of spelling or grammatical errors on the box and on the site, the quality of the packaging, or unusual packaging At the international level, it is essential to consider intensifying international collaboration through coordinated control operations, international cooperation, in particular with the WHO, which does not primarily aim to regulate the sale of medicines on the internet. But it can, however, provide direct assistance to countries and regions to strengthen drug regulation.
CONCLUSIONS
Health products purchased over the internet can cause consumer safety concerns. The health tragedies linked to falsi ied products show that counterfeiting of medical products is not to be taken lightly, as it continues to cause victims throughout the world. Indeed, the traf icking of counterfeit or falsi ied medicines is the work of a chain of actors who are independent of each other but structured among themselves and integrated into international networks. The link is thus made between foreign producers and consumer countries via complex procedures and routes that allow them to avoid customs and health controls on a massive scale. The challenge for health authorities is now to develop an appropriate regulatory approach to control the purchase of these products on the internet and to put in place measures to prevent and minimize the risks associated with this practice. | 2021-04-17T11:34:26.090Z | 2020-12-21T00:00:00.000 | {
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261146462 | pes2o/s2orc | v3-fos-license | 4D‐CT deformable image registration using unsupervised recursive cascaded full‐resolution residual networks
Abstract A novel recursive cascaded full‐resolution residual network (RCFRR‐Net) for abdominal four‐dimensional computed tomography (4D‐CT) image registration was proposed. The entire network was end‐to‐end and trained in the unsupervised approach, which meant that the deformation vector field, which presented the ground truth, was not needed during training. The network was designed by cascading three full‐resolution residual subnetworks with different architectures. The training loss consisted of the image similarity loss and the deformation vector field regularization loss, which were calculated based on the final warped image and the fixed image, allowing all cascades to be trained jointly and perform the progressive registration cooperatively. Extensive network testing was conducted using diverse datasets, including an internal 4D‐CT dataset, a public DIRLAB 4D‐CT dataset, and a 4D cone‐beam CT (4D‐CBCT) dataset. Compared with the iteration‐based demon method and two deep learning‐based methods (VoxelMorph and recursive cascaded network), the RCFRR‐Net achieved consistent and significant gains, which demonstrated that the proposed method had superior performance and generalization capability in medical image registration. The proposed RCFRR‐Net was a promising tool for various clinical applications.
Four-dimensional computed tomography (4D-CT) was referred to as a technique whereby the three-dimensional computed tomography (3D-CT) volume containing a moving structure was imaged over a period, creating a dynamic volume dataset. 1 4D-CT had previously been widely utilized in radiation oncology for planning purposes, especially for tumors located in the thoracic cavity, abdomen, and breast.In recent years, 4D-CT has opened avenues in the diagnostic arena. 2 In the diagnosis of central nervous system diseases, 4D-CT could be utilized to evaluate vascular pathology in the brain and spine.
Additionally, 4D-CT provided the added benefits of perfusion maps and the visualization of structural changes, such as hydrocephalus or hematoma, which could aid in surgical planning.It was superior to current time-resolved MR angiography in terms of both spatial and temporal resolution. 3For cardiac imaging, 4D-CT could be used to visualize the heart and pulmonary vessels and further characterize and measure the in-vivo 3D deformations of both the right ventricular outflow tract and the pulmonary arteries throughout the cardiac cycle. 4formable image registration (DIR) was essential in the clinical application of 4D-CT.DIR was the process of establishing the nonlinear correspondences between an image pair of moving and fixed images.Though DIR had been extensively studied for decades, it remained an active area of research since its current performance did not fully meet the increasingly high accuracy and speed requirements for registration.Furthermore, the large and complex lung motions and low image contrast of abdominal 4D-CT images posed additional difficulties for accurate registration.
The current DIR methods could be divided into two categories: The DVF optimization-based methods included elastic matching, 5,6 statistical parametric mapping, 7 Demons, 8,9 etc. Popular diffeomorphic transform-based methods included large diffeomorphic distance metric mapping (LDDMM), 10,11 diffeomorphic anatomical registration through exponentiated lie algebra (DARTEL), 12 standard symmetric normalization (SyN). 13All these non-DL-based approaches optimized the transformation for each image pair, resulting in slow registration, especially for 3D and 4D images.
5][16][17] According to the output of the network, the deep learning-based DIR methods could be divided into two categories: (1) DL-based similarity calculation; and (2) DLbased DVF prediction.The DL-based similarity calculation method was normally developed for multi-modality image registration.The similarity was normally used by incorporating traditional registration methods for iterative optimization or another DL network.For example, Cheng et al. 18 proposed a DL-based similarity method that trained a binary classifier to learn the correspondence of two image patches from CT and MR image pairs.The classification output was transformed into a continuous probability value, which was then used as the similarity score.
The DL-based DVF prediction method included supervised and unsupervised strategies.For the registration methods based on the supervised learning strategy, the true DVF (i.e., ground truth) calculated based on the moving image and fixed image was used to train the registration network.Three methods were normally used to generate the ground truth: (1) random DVF generation; (2) traditional registration method-based DVF generation; and (3) DVF simulation generation.For the random DVF generation method, the generated DVF was used as the ground truth.The original image (the fixed image) was warped based on the simulated DVF to generate the moving image.
However, the simulated DVF might introduce bias during training since the simulated DVF was unrealistic and quite different from actual physiological motion.For the traditional registration method-based DVF generation method, the traditional registration methods were used to register the image pair to generate the ground truth for registration network training.Using this method, the performance of the developed registration network was greatly affected by the DVF generation methods.Further, finite element (FE) simulations could also be used in DVF generation. 19mpared with the supervised learning strategy-based registration methods, the unsupervised learning strategy-based registration methods only required the image pairs to be registered without the true DVF during model training.In general, the unsupervised learningbased registration methods calculated the DVF using the input image pair and then performed interpolation and deformation on the moving image to generate the warped image. 20,21The unsupervised learning strategy-based registration methods could be classified into three categories, including generative adversarial network (GAN) methods, reinforcement learning (RL) methods, and convolutional neural network (CNN) methods.
The GAN-based methods were mostly used to register the multimodality images by mapping images from one modality to another.Mahapatra et al. 22 trained a GAN to perform registration between retinal color fundus images and fluorescein angiography images.Tanner et al. 23 employed CycleGAN for DIR between MR and CT images.
They firstly transformed the source domain to the target domain before calculating a monomodality image similarity.They discovered this method could achieve, at best, similar performance to the traditional multi-modality deformable registration methods.Further, the accuracy of the absolute intensity mapping of GAN remained to be investigated.The combination of RL and CNN had been applied to decompose the registration task into a sequence of classification problems.This method was mainly employed for rigid registration.
Liao et al. 24 used RL to perform rigid cone-beam CT (CBCT)-CT image registration.Krebs et al. 25 built a statistical deformation model (SDM) for prostate 2D and 3D MR image registration.The method achieved dice similarity coefficient scores of 0.87 and 0.80 for 2D and 3D images, respectively.The unsupervised learning-based CNN methods presented comparable performance to the traditional registration methods recently. 21,26,27The proposition of spatial transformation networks (STN) promoted the development of unsupervised deep learningbased image registration methods since the STN allowed the definition of loss functions without any manually aligned or pre-aligned image pairs. 28de Vos et al. 21applied the proposed STN to the field of image registration based on unsupervised learning for the first time.In this literature, the STN was directly connected to the network, and the generated DVF was used to deform the moving image.During the training process, the deformed and fixed images were used to calculate the training loss, which was back-propagated through the differentiable warping operation and gradually optimized to minimize the value.Balakrishnan et al. 27 presented a fast learning-based registration framework (called VoxelMorph) for medical image registration.In their study, the registration task was formulated as a function that mapped the input image pair to a DVF that aligned the input images.
The function was parameterized via the CNN and optimized based on the input image pairs.The studies of de Vos et al. and Balakrishnan et al. both indicated that the proposed unsupervised methods outperformed state-of-the-art iterative registration methods, while operating a few orders of magnitude faster. 21,27These proposed networks were enforced to make straightforward predictions, which might be a burden when handling complicated deformations, especially with large deformations. 29 Voset et al. 30 proposed a DL image registration (DLIR) framework for unsupervised affine and deformable image registration.DLIR stacked multiple networks into a larger architecture to perform coarse-to-fine image registration.DLIR was trained on each cascade one by one, that was, after fixing the weights of previous cascades.
The volume tweening network (VTN) was an end-to-end cascading scheme that resolved large displacements. 31VTN jointly trained all cascades, while the similarity was measured based on the fixed image and all warped images.Zhao et al. 29
| Recursive cascaded networks
The fixed and moving images were defined over the 3D spatial domain.The work in this paper focused solely on the 3D image volume with grayscale data.Theoretically speaking, the proposed method was dimension-independent.The DVF was the mapping of ϕ : Ω !Ω.
For DIR, a reasonable and plausible DVF should be continuously vary- The entire registration task was cascaded by recursively performing registration on the warped images.The R θ included three cascaded registration subnetworks, denoting as r 1 , r 2 , and r 3 .Following the recursion, the moving image was warped successively three times.
Each cascaded subnetwork was an independent image registration network.The n-th cascaded subnetwork (r n ) could predict the DVF (ϕ n ) based on the input warped image (I nÀ1 w ) and the fixed image, as Equation ( 2).The predicted DVF of the entire network (ϕ RCFRRÀNet ) was the composition of the DVFs of the three cascaded subnetworks: The final warped image (I 3 w ) was generated based on the warped image (I 2 w ) of r 2 and the predicted DVF (ϕ 3 ) of r 3 , which could also be expressed using the input moving image and the final predicted DVF, as: The DVF prediction network was implemented using a ResNetsimilar architecture, the full-resolution residual network (FRRN). 32,33 example of the abstract structure of the FRRN was provided in residual stream and a pooling stream.The residual stream carried feature maps at full resolution with precise boundary information.The features of the residual stream were calculated by adding successive residual units.The pooling stream carried high-level feature maps with information on image texture.The features of the pooling stream were the direct output of the multiple convolution and pooling operations applied to the input images.By fusing the two processing streams, both kinds of features could be calculated simultaneously.
The FRRN was constructed using a sequence of full-resolution residual units (FRRUs). 32The abstract structure of the FRRU was presented in Figure 1c.Since the FRRN could process the residual stream and pooling stream synchronously, each FRRU had two inputs and two outputs accordingly.Let x nÀ1 denote the input of the residual stream to the nth FRRU and y nÀ1 denote the input of the pooling input.The outputs, functions of the residual stream (Fr), and pooling stream (Fp) of the nth FRRU were defined as: where W Fr and W Fp were the parameters of the residual stream and pooling stream, respectively.In Figure 1c, the red box indicated the function of the pooling stream, while the blue box indicated the function of the residual stream.
The FRRU firstly reduced the size of the feature map of the residual stream using a max-pooling layer and then concatenated the two feature maps of the two streams.Next, the concatenated feature maps underwent two 3 Â 3 convolution layers, aiming to extract highly local and complex features.Each convolutional layer was followed by a spatial batch normalization (BN) layer and a rectified linear activation function (ReLU). 33The outputs of the latter convolution layer were fed into the pooling stream and residual stream of the next FRRU.For the pooling stream, the outputs were directly used as inputs.For the residual stream, we adjusted the number of feature channels using a 1 Â 1 convolution layer and upscaled the spatial dimensions using an unpooling layer.
To use the standard gradient-based optimization method, the spatial transformation function-based differentiable operation was used to calculate I m ∘ ϕ: 28 For the voxel v of the I m , the voxel (subpixel) location ϕ v ð Þ was calculated.Since the voxels were only defined at the integer locations, the voxel of I m ∘ ϕ v ð Þ was calculated based on the neighboring voxels using linear interpolation: where principles employed by state-of-the-art methods. 32,34,35The base channel, which was different from Simonyan and He's studies, was set to 32 to have a manageable number of trainable parameters. 34,36The channel number was doubled after each pooling operation (up to the certain maximum number).The encoder and decoder formulations were used according to Noh and Pohlen's studies. 32,35The network architectures of the three cascaded registration subnetworks were presented in Figure 2.
| Loss function
Three cascaded registration subnetworks were jointly trained by merely calculating the unsupervised loss (L us ) between the fixed image and the final warped image. 29This allowed the recursive cascaded networks to learn to perform progressive registration cooperatively.The loss function consisted of two components, which were the image similarity loss (L sim I f ,I w ð Þ) and the DVF regularization loss 27 The image similarity loss penalized the differences in image appearance between I f and I 3 w .The DVF regularization loss penalized the local spatial variations in DVF.
where α indicated the regularization parameter.The α was set as 0.01 according to the previous work of Balakrishnan et al. 27 The L sim I f , I 3 w was conducted using the mean squared error: Minimizing the term L sim I f , I 3 w , would enforce I 3 w to approximate I f .It might generate an unrealistic non-smooth DVF.The DVF regularization loss encouraged a smooth DVF using the diffusion regularizer on the spatial gradients: 2.5 | Experiments
| Dataset
The proposed RCFRR-Net was trained and tested using a retrospective dataset involving 13 patients' 4D-CT images.All images were Each 4D-CBCT dataset also included 10 phases.The 3D images were resampled and normalized prior to testing.The 3D image of the first phase was used as the fixed image, and the 3D images of the other nine phases were used as the moving images.A total of nine CBCT image pairs were included for the 4D-CBCT testing.
| Evaluation
To compare the registration performance of the proposed RCFRR-Net with other DIR methods, we used the iteration-based demon registration method as the first comparison method. 9In addition, two deep learning-based methods, VoxelMorph, and recursive cascaded networks (RC-Net), were also included as comparison methods. 27,29xelMorph and RC-Net were trained using the same training dataset.The VoxelMorph and RC-Net were both constructed based on the vm2double architecture.The hyperparameters of the two networks were consistent with the previous papers. 27,29We developed Root mean square error (RMSE), normalized cross-correlation (NCC), structural similarity index (SSIM), and Dice score were calculated for quantitative evaluations. 37,38Considering that the organs in the abdomen, for example, the liver and stomach, had large amplitudes of motion during the respiratory process, the registration accuracy was also measured based on these key organs.Four regions of interest (ROIs), including the liver, stomach, lung, and surroundings, were selected from the global image to calculate the quantitative metrics for registration evaluation.The ROIs had the same size of 30*30*5.
The ROI-1 and ROI-3 were chosen in the stomach and lung regions.
The ROI-2 and ROI-4 were chosen in the liver and lung regions.The example slices of the ROIs were presented in Figure 3.
The RMSE indicated the squared differences of voxel intensities of two images and was defined as: where i indicated the index of the voxel in the image, m indicated the total number of the voxels in the image, μ i w was the intensity value of the ith voxel in the warped image, and μ i f was the intensity value of the ith voxel in the fixed image.
The NCC was used to measure the correlation between two images: where μ w and μ f were the mean values of the warped image and fixed image, respectively.
The SSIM indicated the similarity of the two images regarding three terms, namely the intensity term, the contrast term, and the structural term.It was proposed to provide a good approximation of perceptual image quality: where were the small constants to avoid instability of SSIM calculation.Here, L was the dynamic range of the input image.σ w and σ f were the standard deviations of the warped image and fixed image, respectively.σ wf was the cross-covariance between the warped and fixed images.
The Dice score was a spatial overlap index.In our study, the Dice score was calculated based on the selected ROIs.The Dice score was measured: where ROI W and ROI F were the ROIs of the warped image and fixed image, respectively.
| Implementation
The proposed algorithm was implemented in Python 3.6 and PyTorch 1.7.0 framework. 39The proposed model was trained using a batch size of 1 on a NVIDIA GeForce RTX 3090 GPU with 24 GB of memory.The training stage ran for 9 Â 10 4 iterations with the Adam gradient optimizer. 40The learning rate was initially 10 À4 and halved after 3 Â 10 4 iterations and again after 6 Â 10 4 iterations.
| Registration evaluation of RCFRR-Net
Table 1 showed the global image and ROI-based comparison of the average registration results using different registration methods over the 10 image pairs of the testing patient.As seen in Table 1, the Vox-elMorph performed better than the demon registration method according to the global image evaluation and performed comparably to the demon registration method according to the ROI-based evaluation.The RC-Net-3 performed better than the VoxelMorph and demon registration methods.The RC-Net-10 had slightly better performance than the RC-Net-3.The RCFRR-Net showed optimal performance among all registration methods.Figure 3 showed the axial, coronal, and sagittal planes and selected ROIs of the moving images, fixed images, and warped images registered using different methods.
The absolute difference between the fixed images and the warped images of different registration methods were shown in
The percentage between the RMSE difference and the RMSE before registration was 73.46%, 10.95%, and 2.4%, respectively.The RMSE difference and percentage decreased significantly with the increase in the number of cascaded networks.This trend could also be observed in the comparison based on ROIs.
| Registration runtime
Table 4 showed the runtime results of VoxelMorph, RC-Net-3, RC-Net-10, and RCFRR-Net.Since the demon registration method required a few minutes to finish the registration, we only reported the runtimes of the DL-based methods in this study.All methods were conducted using the GPU.The VoxelMorph computed a registration with an average runtime of 1.334 ± 0.0219 s.
For the VoxelMorph method, the reported runtime was 0.45 s, while it had an average runtime of 1.334 ± 0.0219 s in this study. 27is might result from the difference in the datasets.The dataset for this study was abdominal 4D-CT images, and the previous
| Registration evaluation on DIRLAB datasets
Table 5 showed the global image and the ROI-based comparison of the average RMSE, NCC, SSIM, and Dice score using different registration methods for the testing patients from DIRLAB datasets.Consistent with the testing dataset from our institution, the RCFRR-Net showed optimal performance among all registration methods.Figure 8 presented the axial, coronal, and sagittal planes and selected ROIs of the moving images, fixed images, and warped images registered using different methods using images from DIRLAB datasets.The absolute difference between the fixed images and the warped images of different registration methods were shown in Figure 9.As shown in the residual images, the RCFRR-Net showed the smallest difference.
Using the quantitative metrics for the registration evaluation, the average RMSE between the moving image and the fixed image was 78.66 ± 13.04 HU while decreasing to 15.54 ± 2.47 HU between the warped image and the fixed image after the proposed RCFRR-Net registration.The NCC increased from 0.9835 ± 0.0050 to 0.9997 ± 0.0001 and the SSIM increased from 0.7326 ± 0.0430 to 0.8427 ± 0.0460 using the RCFRR-Net registration.
For the comparison based on ROIs, the RMSE was 336.00 ± 40.The novelty of this study could be summarized in three folds.
First, the RCFRR-Net was trained using the unsupervised approach.
The advantage of unsupervised training was that it could alleviate the problem of a lack of ground truth DVFs.Since ground truth DVF was not required, any 4D-CT dataset could be used for network training.
Second, the RCFRR-Net was constructed using the recursive cascaded method.The similarity was only measured based on the final warped
( 1 )
the non-deep learning (DL)-based methods; and (2) the DL-based methods.Popular non-DL-based registration methods included two categories: the deformation vector field (DVF) optimizationbased methods and the diffeomorphic transform-based methods.
presented a general recursive cascaded networks (RC-Net) architecture that enabled learning deep cascades for deformable image registration.The registration procedure was recursive, such that every cascade learned to perform a progressive deformation for the current warped image.The similarity was only measured based on the fixed image and the final warped image.The base network of the RC-Net had a simple and identical architecture of VoxelMorph and VTN.In addition, the base network had the same network depth and parameters.One drawback of the RC-Net was that the number of parameters increased linearly with the number of stacks.The RC-Net with five cascades showed a Dice value of 0.949.The RC-Net achieved a Dice value of 0.953 to 0.956 from 10 to 20 cascades using the VTN base network.With a greater number of cascades, the improvement in registration performance might be limited.This article made several technical contributions towards the goal of developing neural networks for abdominal 4D-CT image registration.(i) We introduced a novel registration network with three recursive cascaded subnetworks to perform progressive registration.Each subnetwork was designed with different architectures to capture different movement conditions.(ii) We proposed to calculate the DVF based on the full-resolution residual network, which could capture the precise boundary and texture information of the image simultaneously.(iii) We evaluated the proposed network using multiple datasets (an internal 4D-CT dataset, a public DIRLAB 4D-CT dataset, and a 4D-CBCT dataset).
Figure
Figure 1a depicted the workflow of the proposed recursive cascaded full-resolution residual network (RCFRR-Net).Considering the large and complex motion patterns of abdominal 4D-CT images, the recursive cascaded method was used to conduct successive registration.The RCFRR-Net had three independent registration subnetworks with different network parameters and depths, aiming to perform global and refined registrations.Each subnetwork was directly connected and used as an independent network with respective input and output.The first subnetwork used the fixed and moving image pairs as inputs.The second and third subnetworks used the fixed image and the warped image (output of the front registration subnetwork) as the paired input images.To accurately capture the precise boundary information of image elements, the subnetwork was developed by merging a full-resolution residual network (FRRN) and a spatial transformation function.The loss of the RCFRR-Net was calculated based on the fixed image and the final warped images during the network training process.The trained network was applied to the input image data to calculate the final warped image to achieve registration.
ing.A DVF prediction function, R θ I f ,I m ð Þ, was developed in the proposed method, where θ indicated the network parameters, I f indicated the fixed images and I m indicated the moving images.The function R θ used the I f , I m as inputs and predicted the DVF (ϕ) and the warped image I w as outputs.The I w was generated based on the predicted DVF and the moving image:
2. 3 |
Registration subnetworks Each cascaded registration subnetwork consisted of a DVF prediction network and a spatial transformation function.Each subnetwork was designed to predict a DVF and a warped image of itself based on the input moving image (or warped image from the front registration subnetwork) and the fixed image.The DVF prediction network was designed to compute the DVF.The spatial transformation function was used to warp the input moving image based on the predicted DVF.
Figure 1b .
Figure 1b.The FRRN processed two distinct feature streams: a Since the spatial transformation function was differentiable, the errors could be backpropagated during the training process.The three cascade registration subnetworks were designed with different network parameters and depths in this study.The top (first) cascade was designed to learn global registration with a shallow network architecture (FRRU blocks: 5, maximum number of FRRU channels: 128).The second cascade was designed with a deeper network architecture compared with the first cascade (FRRU blocks: 7, maximum number of FRRU channels: 256).The bottom cascade was designed to learn the refined registration with the deepest network architecture (FRRU blocks: 9, maximum number of FRRU channels: 512).The proposed architectures were constructed based on obtained from Siemens SOMATOM Definition AS using 120 kVp tube voltages.The original image pixels of the datasets ranged from 0.6836 to 0.9736 mm.The slice thickness ranged from 2 to 3 mm.All images were cropped and resampled to the same size of 96 Â 96 Â 96.Image gray values were normalized to [0, 1].Each 4D-CT dataset included 11 phases of 3D-CT images throughout the respiratory cycle: one initial phase (denoted as: In0%), five exhalation phases (denoted as: Ex20%, Ex40%, Ex60%, Ex80%, and Ex100%), and five inhalation phases (denoted as: In20%, In40%, In60%, In80%, and In100%).The 4D-CT datasets of 12 patients, including a total of 132 3D-CT images, were used as the training dataset.During training, image pairs between any two phases of the 11 phases were used as the moving image and the fixed image.In total, the training dataset included 1320 pairs of 3D-CT images.For testing, the 3D-CT image of the exhale and inhale phases was registered to the initial phase image.A total of 10 CT image pairs were used for testing.
F I G U R E 2
The network architectures of the three cascade registration subnetworks.The notation Conv (n  n, m) indicated a convolution layer of m kernels with each of size n  n.The notations RU(v) and FRRU(v) indicated the RU and FRRU whose convolutions included v channels.The three cascade registration subnetworks differed in network depth.(a) Subnetwork-1; (b) Subnetwork-2; and (c) Subnetwork-3.The performance of the proposed RCFRR-Net was tested using the public DIRLAB dataset (www.dir-lab.com).The 3D images were resampled to the same size of 96  96  96 and normalized to the same range of 0 to 1.Each 4D-CT of the DIRLAB dataset included 10 phases.The 3D image of the first phase was used as the fixed image, while the images of the other nine phases were used as the moving images.Nine image pairs were generated for each case.Three cases (case 1, case 2, and case 3) from DIRLAB were included.Thus, a total of 27 image pairs were used for testing based on the DIRLAB dataset.Furthermore, to test the generalization capability of the proposed RCFRR-Net, the authors tested the proposed RCFRR-Net using 4D-CBCT images.The images were obtained from "The Sparse-view Reconstruction Challenge for Four-dimensional Cone-beam CT" (https:// image-x.sydney.edu.au/spare-challenge/).The 4D-CBCT images had higher noise and artifact levels compared with the 4D-CT images.
Comparison of the moving images, fixed images, and warped images of different registration methods.The first three rows showed the comparison of global images, and the last two rows showed the comparison of ROI-1 and ROI-2.The red box in the axial plane and the line in the coronal plane indicated the example slices of ROIs.The red arrow in the figures indicated the organs with large motion.The images of the six columns were the fixed images, moving images, and warped images of demon-based registration, VoxelMorph, RC-Net-3, RC-Net-10, and RCFRR-Net, respectively.Display window: [-200, 300] HU. two RC-Nets with three and 10 cascades, abbreviated as RC-Net-3 and RC-Net-10, respectively.
Figure 4 .
It could be seen from the residual images that the difference images from the proposed RCFRR-Net were smaller compared with the comparison methods, especially in the selected ROIs.Using the quantitative metrics for the registration evaluation, the original average RMSE between the moving image and fixed image was 82.52 ± 23.75 HU.With the proposed RCFRR-Net, the average RMSE between the warped image and fixed image after the registration decreased to 10.88 ± 3.78 HU.The NCC increased from 0.9831 ± 0
F I G U R E 6
Comparison of the residual images between the fixed images and warped images of intermediate cascades.The first three rows showed the comparison of global images, and the last two rows showed the comparison of ROI-1 and ROI-2.The images of the four columns were the residual images between fixed images and moving images, and warped images of Cascade-1, Cascade-2, and Cascade-3, respectively.Display window: [0, 100] HU.ROIs based on the warped image and fixed image after the registration using RCFRR-Net.For the selected ROIs of the moving image and the fixed image, the ROI-based NCC and SSIM were lower than the values based on the overall image.The NCC increased from 0.6621 ± 0.1102 to 0.9991 ± 0.0002 for the ROI-1 and from 0.7937 ± 0.0795 to 0.9993 ± 0.0001 for the ROI-2.The SSIM also increased from 0.3504 ± 0.1171 to 0.9695 ± 0.0091 for the ROI-1 and from 0.4343 ± 0.1175 to 0.9503 ± 0.0183 for the ROI-2.The Dice score increased from 0.5532 ± 0.0766 to 0.8681 ± 0.0201 for the ROI-1 and from 0.7836 ± 0.0464 to 0.9579 ± 0.0111 for the ROI-2.Furthermore, to access the progressive registration process of the RCFRR-Net, the intermediate registration results of the RCFRR-Net were also evaluated, that was, the registration results of the first cascaded network (Cascade-1) and the second cascaded network (Cascade-2).
3. 2 |
Registration evaluation of RCFRR-Net with different number of cascadesTo measure the registration results of RCFRR-Net with different number of cascades, we constructed RCFRR-Net with two cascaded registration subnetworks and one registration subnetwork.The maximal number of cascades was three in the current study to consider the trade-offs between registration time and accuracy.In this section, the RCFRR-Net with three cascades, two cascades, and one subnetwork were denoted as RCFRR-Net-3, RCFRR-Net-2, and RCFRR-Net-1, respectively.The network architecture and hyper-parameters of the RCFRR-Net-2 were consistent with the second and third subnetworks of the RCFRR-Net-3.The network architecture and hyper-parameters of the RCFRR-Net-1 were consistent with the third subnetwork of the RCFRR-Net-3.
study was developed for brain MR datasets.Compared with the brain dataset, 4D-CT images had more complex deformations.The runtime of the RC-Net-3 was slightly longer (1.446 ± 0.0207 s) than that of the VoxelMorph.The RC-Net-10 (1.878 ± 0.0216 s) had a longer runtime than the RC-Net-3.Since the subnetworks of the RCFRR-Net were relatively deeper than the RC-Net, the runtime of the RCFRR-Net-3 was slightly longer than the RC-Net-10 by 1.940 ± 0.0158 s.The runtimes of the RCFRR-Net-1, RCFRR-Net-2, and RCFRR-Net-3 increased gradually.T A B L E 5 Comparison of RMSE, NCC, SSIM, and dice score using different registration methods based on DIRLAB datasets.
The RMSE values for the selected CBCT ROIs were higher than the RMSE values of the selected CT ROIs using the RCFRR-Net registration.For the selected ROIs of the moving image and the fixed image, the RMSE decreased from 296.25 ± 97.10 HU to 57.80 ± 13.01 HU, from 282.10 ± 100.88 HU to 55.23 ± 14.42 HU, from 282.68 ± 102.69 HU to 56.25 ± 12.51 HU, and from 254.09 ± 81.85 HU to 54.91 ± 13.42 HU, for ROI-1, ROI-2, ROI-3, and ROI-4, respectively.The NCC, SSIM, and Dice score all had the highest values for the selected ROIs.4 | DISCUSSIONIn this study, we proposed a novel unsupervised deep learning-based method for abdominal 4D-CT image registration.The developed method was tested using multiple datasets (an internal 4D-CT dataset, a public DIRLAB 4D-CT dataset, and a 4D-CBCT dataset).The RCFRR-Net was trained using the internal 4D-CT dataset without the DIRLAB 4D-CT dataset or 4D-CBCT dataset.The RCFRR-Net outperformed the comparison methods using all testing datasets.This was a shred of strong evidence that the RCFRR-Net had a high generalization capability in medical image registration.
F I G U R E 1 0
Comparison of the moving images, fixed images, and warped images of different registration methods on 4D-CBCT datasets.The red dotted lines indicated the organs with large motion.Display window: [À800, 200] HU. image and the fixed image, allowing all cascades to be trained jointly and perform the progressive registration cooperatively.Third, the cascaded registration subnetwork was developed based on a novel ResNet-similar architecture, the full-resolution residual network.The FRRN processed two distinct feature streams: a residual stream and a pooling stream.The residual stream carried feature maps at full resolution with precise boundary information.The pooling stream carried high-level feature maps with information on image texture.By fusing the two processing streams, both kinds of features carrying image boundary and element information could be obtained simultaneously.Although the proposed RCFRR-Net could conduct 4D-CT image registration with high speed and accuracy, it still needed improvement in future work.The RCFRR-Net was trained based on the global image, which has a relatively small size.The 3D images were resampled to a size of 96 Â 96 Â 96.The RCFRR-Net was not trained using image patches.Because the image patches were usually randomly cropped from the global image, there might be differences in the organizational structure within the paired image patches.This might introduce inevitable errors during the model training process.In the future, we will design an advanced and more appropriate patch extraction method for registration network training.Without increasing the computation resource, the proposed method could achieve image registration at larger sizes.Since the RCFRR-Net was trained in a completely unsupervised manner without any prior knowledge about the ground truth, the loss was essential for accurate DVF predictions.In the current study, the loss was composed of similarity in the image domain and smoothness in the DVF domain.The loss was calculated equally for different regions of the global image without considering the physiological motion variance of different regions.In future studies, the physiological motion around the lung could be specifically modeled to accurately capture the complex motion conditions to improve the DIR's performance.For example, the adaptive weights for image similarity and DVF regularization could be incorporated into the loss function.5| CONCLUSIONSThis study presented an unsupervised recursive cascaded architecture for abdominal 4D-CT DIR.Experiments based on diverse datasets and evaluation metrics demonstrated that the proposed architecture achieved significant gains over state-of-the-art methods.With the superiority of outstanding performance and the general applicability of the unsupervised manner, we expected that the proposed architecture could potentially be extended to more DIR clinical tasks.AUTHOR CONTRIBUTIONSLei Xu: Formal analysis (lead); investigation (lead); methodology (lead); resources (lead); software (lead); validation (lead); visualization (lead); writingoriginal draft (lead); writingreview and editing (lead).Ping Jiang: Writingreview and editing (supporting).Tiffany Tsui: F I G U R E 1 1 Comparison of the residual images between the fixed images and warped images of different registration methods on 4D-CBCT datasets.Display window: [0, 400] HU.
1
Comparison of RMSE, NCC, SSIM, and dice score using different registration methods.
T A B L E 2 Comparison of RMSE, NCC, SSIM, and Dice score of intermediate cascades.
Table 2
presented the results of intermediate cascades based on the global image and ROIs.As shown in the table, the RCFRR-Net achieved performance gains with the higher cascade number.Figure 5 showed the axial, coronal, and sagittal planes and 30.50 ± 4.02, and 17.79 ± 2.85 for the ROI-1 based on Cascade-1, Cascade-2, and Cascade-3, respectively.The RMSE decreased from 261.36 ± 71.35 (before registration) to 83.91 ± 17.32, F I G U R E 7 Box plots for the results of intermediate cascades.RMSE (a, b, c), NCC (d, e, f) and SSIM (g, h, i) comparison of intermediate cascades based on the global image, ROI-1, and ROI-2.24.18 ± 2.31, and 14.58 ± 2.28 for the ROI-2 based on Cascade-1, Cascade-2, and Cascade-3, respectively.The NCC, SSIM, and Dice score all gradually increased with higher cascade numbers for both ROIs. Figure 7 plotted the results of intermediate cascades to illustrate progressive registration.
Table 3
Comparison of the RMSE, NCC, SSIM, and Dice score using RCFRR-Net with different number of cascades.
presented the global image and ROI-based comparison of the RMSE, NCC, and SSIM of RCFRR-Net with different number of cascades.As shown in the table, RCFRR-Net-3 outperformed RCFRR-Net-1 and RCFRR-Net-2 as expected.For the global T A B L E 3 Comparison of RMSE, NCC, SSIM, and Dice score using different registration methods based on 4D-CBCT datasets.
ROI-1 and from 0.5497 ± 0.086 to 0.9184 ± 0.0177 for the ROI-2.T A B L E 6
Table 6
showed the global image and ROI-based comparison of the average RMSE, NCC, SSIM, and Dice score using different registration methods for the 4D-CBCT testing patient.Due to the high level of image noise and artifacts in CBCT images, the registration accuracy was worse than the registration accuracy of CT images.As could be seen from | 2023-08-26T15:34:56.577Z | 2023-08-22T00:00:00.000 | {
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118906430 | pes2o/s2orc | v3-fos-license | BFKL Physics in Dijet Production at the LHC
The production in hadron-hadron collisions of jet pairs with large rapidity separation and comparable modest transverse momentum is, in principle, described by the perturbative QCD BFKL equation. The measurement of such jet pairs appears well suited to the LHC detectors, with their ability to detect forward jets. We present predictions for dijet cross sections and correlations obtained using a BFKL Monte Carlo which allows kinematic and other subleading effects to be incorporated. The enhanced phase space for gluon emission at the LHC makes the BFKL behavior somewhat easier to observe than at the Tevatron, although kinematic effects are still important. The production of forward jets in association with heavy Higgs bosons via the gauge boson fusion mechanism is also studied and compared with QCD dijet production.
Introduction
In perturbative QCD physical quantities are typically computed in fixed-order expansions in powers of the coupling constant α S . In some kinematic regimes, however, large logarithms multiply the coupling and the series must be resummed to all orders. In certain cases this resummation is possible and meaningful predictions can still be obtained. For example, large logarithms in the small-x region in deep inelastic lepton-hadron scattering are resummed by the Balitsky, Fadin, Kuraev and Lipatov (BFKL) equation [1], to yield the well-known BFKL prediction F 2 ∼ (1/x) λ , with λ = 4C A ln 2 α S /π ≈ 0.5.
In hadron collisions BFKL resummation also applies to production of jet pairs at large rapidity separation ∆ when the jets have comparable transverse momenta that are small compared to the center-of-mass energy. At large values of ∆, the cross section receives contributions from real and virtual gluons emitted in the rapidity interval between the two jets, as illustrated in Fig. 1(a). The BFKL equation resums the leading logarithmic contributions from these gluons [2], giving rise to a cross section that increases with ∆,σ ∼ exp(λ∆), with λ defined above. Mueller and Navelet [2] pointed out the potential of this increase, which does not appear in lowest-order QCD, to provide a way to investigate BFKL physics at hadron colliders by looking at dijet production at large rapidity separation.
Unfortunately the ∆ increase disappears when the subprocess cross section is folded in with parton distribution functions (pdfs), which decrease with ∆ more rapidly thanσ increases. Several methods have been suggested to avoid this pdf sensitivity when studying 'Mueller-Navelet' jets. One [3,4,5] is to study the decorrelation in the two jets' azimuthal angles that arises from emission of gluons between the jets; BFKL predicts a stronger decorrelation than does fixed-order QCD, and this prediction should be relatively insensitive to the pdfs. Another [2,6] is to look for the ∆ increase in the ratio of dijet cross sections at two center-of-mass energies for corresponding parton momentum fractions, so that the pdfs cancel.
In practice it is not useful to compare analytic asymptotic BFKL predictions directly with experiment because nonleading corrections can be large. In particular, in the analytic BFKL calculation gluons can be emitted arbitrarily, with no kinematic cost, and energy and momentum are not conserved. In Refs. [5,6] (see also [7]) a Monte Carlo approach is used in which the emitted gluons are subject to kinematic constraints, and other nonleading effects such as the running of α S are included. Kinematic constraints are seen to have a significant effect, suppressing the emission of large numbers of energetic gluons. The studies in [5] and [6] focused on the azimuthal decorrelation and cross section ratios at the Fermilab Tevatron pp collider.
Here we are concerned with dijet production at the CERN LHC pp collider. The phase space available for studying dijet production is larger at the LHC than at the Tevatron, because the center-of-mass energy is much higher while measurable jet transverse momentum thresholds increase only slightly, if at all. As a result we expect kinematic constraints to have weaker effects at the LHC, so that the predictions of naive BFKL should be more robust than at the Tevatron. We will address this in more detail below.
BFKL physics (or, more generically, QCD) is not the only potential source of jet pairs with large rapidity separation at the LHC: heavy Higgs production via gauge boson fusion in the process qq → qqH (see Fig. 1(b)) is well known to give rise to forward jets [8,9]. In fact the forward calorimetry in the LHC detectors has been designed to be able to detect jets at large rapidity for the purpose of observing this process [10]. Detection of jets with rapidity |y| < ∼ 5 (i.e. ∆ < ∼ 10) and transverse momentum p T > O(20 GeV) should be possible. In contrast to the BFKL case, where the region between the jets is populated by gluons, the jets produced in association with the Higgs are characterized by an absence of hadronic activity between them -a 'rapidity gap. ' We will see other differences when we compare other features of dijet production in the two cases below. The Higgs process therefore acts as a useful comparator for the BFKL study.
The paper is organized as follows. In the next section we summarize the essential features of the BFKL MC calculation used to obtain our results. In section 3 we present results for dijet production in pp collisions at center-of-mass energy 14 TeV, first in the BFKL approach, and then for jets produced in association with a heavy Higgs boson. In section 4 we investigate the collision energy dependence of the BFKL dijet cross section and present the ratio of cross sections at 10 and 14 TeV. In section 5 we present our conclusions.
2 Improved BFKL approach to dijet production in hadron collisions The differential subprocess cross section for production of a jet pair with rapidity difference ∆ ≡ |y 1 −y 2 | and relatively small transverse momenta p T 1 , p T 2 is given in the BFKL approach by dσ The Laplace transform of the function f with respect to ∆ satisfies the BFKL equation. This equation contains integrals over both real and virtual gluons, and when its Laplacetransformed solution is substituted into Eq. (1), it gives the sum over emitted gluons and total cross section behavior discussed above. This is the 'naive' BFKL case. As indicated in the introduction, the price of having an analytic solution is integration over radiated gluons with arbitrarily large transverse energies. We can improve on this approach by unfolding the implicit sum over gluons that appears in the analytic solution. Details can be found in [5]; see also [7] for a similar approach. The basic idea is that real gluons are separated into 'resolved' and 'unresolved' gluons, the latter being those that fall below a transverse momentum threshold µ 0 and are undetectable as jets. The unresolved gluon contributions are then combined with those from virtual contributions to give an overall form factor that suppresses the cross section. The cross section is then an explicit sum over resolved real gluons; for fixed α S it becomes with R (0) = δ( p T 1 + p T 2 ) and R (n) for n > 0 given in [5], where the expressions for running α S can also be found. The analytic result for the total cross section is reproduced in the limit µ 0 → 0. 1 It is straightforward to implement this in a Monte Carlo program where kinematic cuts can be applied directly. The total cross section is obtained by folding in the subprocess cross section (2) with the appropriate parton distributions functions; see [5] for details.
3 Jet pair production in pp collisions at √ s = 14 TeV
BFKL calculation of dijet production
The results shown here were obtained using the BFKL Monte Carlo program described above and in Ref. [5]. We consider dijet production in which the jets have equal and opposite rapidity y 1 = −y 2 = ∆/2 and transverse momentum greater than some value P T . 2 For the LHC we consider transverse momentum thresholds P T = 20 GeV and P T = 50 GeV. Given the high level of hadronic activity at the LHC, the former value may be slightly optimistic but the latter is certainly achievable [10]. We use CTEQ4L parton distributions [11]. Figure 2 shows the dijet production cross section at the LHC with center-of-mass energy 14 TeV for p T > 20 GeV (upper curves) and p T > 50 GeV (lower curves). All of the curves fall off with increasing ∆ as a result of pdf suppression; note that the difference in momentum thresholds causes the p T > 50 GeV cross sections to reach the kinematic limit at smaller values of ∆ than the p T > 20 GeV curves.
The naive BFKL prediction, obtained using the analytic (asymptotic) BFKL solution combined with pdfs assuming lowest-order kinematics, is shown as dashed curves in the figure. For both p T thresholds, the naive BFKL cross section is largest for all values of ∆; this reflects the rise with ∆ of the subprocess cross section. The falloff with ∆ is due to the pdfs. The BFKL MC results, shown as solid curves, lie well below naive BFKL as a result of the kinematic suppression. 3 Clearly nonleading effects due to kinematics are important. Also shown for comparison are the cross sections in the asymptotic (large ∆) lowest-order QCD limit (see [6] for details about this limit). It lies below the naive BFKL curves, because the subprocess cross section is asymptotically flat in ∆, and it lies above the BFKL MC curves because it reflects only leading-order kinematics, whereas the BFKL MC cross section pays the pdf penalty for emitted gluons.
A characteristic of BFKL dijets that clearly distinguishes them from those in leadingorder QCD is a decorrelation in azimuthal angle at large values of ∆ [3,4,5]. In QCD at lowest order the two jets are strictly back-to-back in the transverse plane, so that their azimuthal angles are completely correlated, independent of rapidity. In contrast, in the BFKL case gluons are radiated in the rapidity interval between the two jets, and as this interval increases more gluons can be radiated. The presence of these gluons weakens the azimuthal correlation between the two jets of interest, so that one sees an increasing azimuthal decorrelation as ∆ increases.
As discussed in [5], applying kinematic constraints as in the improved BFKL MC gives less of a decorrelation than predicted in naive BFKL because emission of arbitrarily many gluons of arbitrary energy is no longer allowed. In fact, for any given transverse momentum threshold, there is some ∆ at which the jet pair alone saturates the kinematic limit, and emission of additional (real) gluons is completely suppressed and the azimuthal correlation returns. As we approach that limiting value of ∆ we therefore expect to see a transition back towards correlated jets. This is illustrated in Fig. 3, where we show the azimuthal decorrelation in dijet production in the improved BFKL MC approach (upper curves). We show the mean value of cos ∆φ where we have defined so that ∆φ = 0 when the two jets are back-to-back in the transverse plane. The jets are completely correlated at ∆ = 0, and as ∆ increases we see the characteristic BFKL decorrelation, followed by a flattening out and then an increase in cos ∆φ as we approach the kinematic limit. Not surprisingly, the kinematic constraints have a much stronger effect when the p T threshold is set at 50 GeV (dashed curve) than at 20 GeV (solid curve); in the latter case more phase space is available to radiate gluons. We also show for comparison the decorrelation for dijet production at the Tevatron for p T > 20 GeV. There we see that the lower collision energy (1.8 TeV) limits the allowed rapidity difference and substantially suppresses the decorrelation at large ∆. The larger center-of-mass energy compared to transverse momentum threshold at the LHC would seem to give it a significant advantage as far as observing BFKL effects is concerned. The lower set of curves in Fig. 3 refer to Higgs production and will be discussed below.
Forward jets from Higgs production via boson fusion
At the LHC, Higgs bosons can be produced in the process qq → qqH, where W or Z bosons emitted by the initial state quarks annihilate to form a Higgs [8], see Fig. 1(b). This mechanism may be important for discovering a heavy M H ∼ 1 TeV Higgs, since the rate there is comparable to the rate from the 'standard' gg → H process, see for example Ref. [12]. For lighter Higgs bosons, weak boson fusion can provide a complementary method of checking that the couplings to heavy quarks and weak bosons are related in the way specified by the Standard Model. Our interest here, however, is in the final-state quark jets accompanying the Higgs. The ability to 'tag' these jets [9] is necessary to suppress backgrounds, for example from qq → W W, ZZ, and tt production. It is the t-channel W and Z propagators (see Fig. 1(b)) that to a large extent determine the regions of phase space populated by the tag jets. Typically, they have O(TeV) energies and transverse momenta less than M W . They therefore naturally have large (roughly equal and opposite) rapidities.
Higgs production via W W and ZZ fusion therefore automatically provides a 'BFKLlike' dijet sample with large rapidity separation. Figure 4 shows the differential cross section d 2 σ H /dy 1 dy 2 (y 1 = −y 2 = ∆/2) as a function of ∆, for M H = 500 GeV and p T > 20, 50 GeV at the LHC. Notice the difference in shape compared to the QCD LO dijet distributions shown in Fig. 2; the tag jets are naturally produced with a sizeable rapidity separation.
The big difference between Higgs and QCD dijet production is of course the t-channel color singlet exchange property of the former. This gives rise to a rapidity gap, rather than a region between the jets populated by BFKL soft gluons, Fig. 1(a). 4 The large rapidity separation decorrelation between the forward jets induced by multigluon emission between them is therefore absent. Interestingly, however, for M H ≫ p T (jet) the centrally produced Higgs boson acts as a jet decorrelator, i.e. the qq → Hqq matrix element does not depend on the relative orientation of p T 1 and p T 2 . Indeed the only significant correlation occurs when the tag jets are produced centrally (small ∆) and all invariants p i · p j are large and of the same order. Three-body kinematics then yields approximately back-to-back jets. The situation is summarised in Fig. 3, which shows the ∆ dependence of cos ∆φ for the qq → qqH process (lower set of curves). We have again taken M H = 500 GeV, but in fact the dependence on the (heavy) Higgs mass is very weak. Evidently the jets accompanying Higgs production are much more decorrelated in azimuth than the Mueller-Navelet QCD jets. In principle, therefore, the Higgs tag jets could act as a benchmark for the decorrelation, since the theoretical predictions shown in Fig. 3 should be reliable.
Collision energy dependence of dijet production at the LHC
The BFKL increase in the dijet subprocess cross section with ∆ is swamped by the decreasing parton distribution functions, as can be seen in Fig. 2. One way [2,6] to account for this is to take a ratio of cross sections at different ∆ but at the same values of parton momentum fraction x so that the pdf dependence cancels out. This requires (at least) two center-of-mass energies. The difficulty with this method, as discussed in [6], is that the cancellation of pdfs only occurs using lowest-order kinematics; like any other quantity, the cross section ratio is subject to corrections due to kinematic effects. As shown in [6], these kinematic effects turn out to be very important at the Tevatron (for √ s = 630 and 1800 GeV) and the improved BFKL MC prediction differs qualitatively from that of naive BFKL. In this section we investigate the dijet cross section ratio at the LHC, for the design center-of-mass energy √ s = 14 TeV and for √ s = 10 TeV, which has been mentioned as a possible first step toward the design energy. At the LHC, the higher center-of-mass energy compared to transverse momentum threshold may mitigate the strong kinematic suppression that spoiled the naive BFKL ratio at the Tevatron. We consider a cross section ratio R 12 at center-of-mass energies √ s 1 and √ s 2 measured at fixed values of rapidity difference ∆ 1 and ∆ 2 chosen so that the asymptotic lowest-order QCD cross sections (including pdfs) are equal [6]. Given ∆ 1 , ∆ 2 is given by The cross section ratio to be measured is then One advantage of this definition is that it uses the rapidity differences between the two outside jets, rather than the parton momentum fractions for the event, which can be more difficult to measure [14].
In Fig. 5 we show R 12 at the LHC with √ s 1 = 10 TeV and √ s 1 = 14 TeV as a function of ∆ 1 , with ∆ 2 determined by Eq. (4). Note that although we show results for all values of ∆, it is really only at the larger values that we expect BFKL behavior to be manifest. In asymptotic LO QCD R 12 = 1 by construction; it is shown for reference as a dotted line in the figure. We show the naive BFKL (lower curves) and improved BFKL MC (upper curves) ratios for p T > 50 GeV (dashed curves) and p T > 20 GeV (solid curves). As at the Tevatron, we see that the BFKL MC predictions deviate strongly from those of naive BFKL due to nonleading effects. The situation is slightly better than at the Tevatron, where the cross section was never larger at 1800 GeV than at 630 GeV, in marked contrast to the naive BFKL expectation. Again, the LHC benefits from the larger available phase space.
Conclusions
In summary, the ability of the LHC general purpose detectors to measure 'Mueller-Navelet' forward jets with large rapidity and modest transverse momentum will allow an important test of QCD 'BFKL' physics. In this paper we have presented the results of an improved BFKL Monte Carlo calculation for dijet production at large rapidity separation at the LHC. In this approach, subleading effects such as kinematic constraints and the running of the strong coupling constant α S are incorporated by unfolding the implicit BFKL sum over emitted gluons to make the sum explicit. 5 We found that, as at the Tevatron [5,6], these subleading effects can be substantial and in particular lead to some suppression of gluon emission. This kinematic suppression is however not as dramatic at the LHC, because of its relatively higher center-of-mass energy compared to the jet transverse momentum threshold. As a result, improved BFKL MC predictions tend to retain more BFKL-type behavior because of the greater phase space for emitting gluons. We saw that as the p T threshold was lowered, the BFKL MC results became more naive-BFKL-like. This was true for both the azimuthal decorrelation and the cross section ratio at different collision energies.
Noting that forward jets are also produced at the LHC in heavy Higgs boson production via gauge boson fusion, we compared the two dijet production mechanisms. The Higgs case is characterized by a lack of hadronic activity in the rapidity region between the two jets (the 'rapidity gap'), in contrast to the BFKL case where the region between the jets is populated by emitted gluons which give rise to an azimuthal decorrelation of the outside jets. Interestingly, however, we found that the Higgs boson itself acts as a decorrelator and 5 Another potentially important effect, not included in the present study, may come from the next-toleading order perturbative corrections to the BFKL equation, which have recently been calculated [15]. the jets produced in association with a heavy Higgs show a stronger decorrelation than the BFKL jets.
The LHC holds promise for studies of dijet production in BFKL physics, and such studies will benefit from jet detection capabilities that extend far into the forward region and allow for p T thresholds as low as possible. However, kinematic effects can still be of quantitative importance, and should therefore be incorporated in the theoretical analyses. | 2019-04-14T02:45:59.279Z | 1998-06-15T00:00:00.000 | {
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16473834 | pes2o/s2orc | v3-fos-license | An Important Role of α-Hemolysin in Extracellular Vesicles on the Development of Atopic Dermatitis Induced by Staphylococcus aureus
Skin barrier disruption and dermal inflammation are key phenotypes of atopic dermatitis (AD). Staphylococcus aureus secretes extracellular vesicles (EVs), which are involved in AD pathogenesis. Here, we evaluated the role of EVs-associated α-hemolysin derived from S. aureus in AD pathogenesis. α-hemolysin production from S. aureus was detected using western blot analyses. The cytotoxic activity of α-hemolysin on HaCaT keratinocytes was evaluated by measuring cell viability after treating cells with soluble and EVs-associated α-hemolysin. To determine the type of cell death, HaCaT keratinocytes were stained with annexin V and 7-AAD. The in vivo effects of α-hemolysin were evaluated by application of soluble and EV-associated α-hemolysin on the mouse skin. The present study showed that increased α-hemolysin was produced by S. aureus colonized on AD patients compared to healthy subjects. α-hemolysin production was also related to AD severity. In addition, EV-associated α-hemolysin was more cytotoxic to HaCaT keratinocytes than soluble α-hemolysin, and α-hemolysin-negative EVs did not induce keratinocyte death. EV-associated α-hemolysin induced necrosis, but soluble α-hemolysin induced apoptosis of keratinocytes. In vivo, skin barrier disruption and epidermal hyperplasia were induced by soluble and EV-associated α-hemolysin. However, AD-like dermal inflammation was only caused by EV-associated α-hemolysin. Moreover, neither skin barrier disruption nor AD-like skin inflammation was induced by α-hemolysin-negative EVs. Taken together, α-Hemolysin secreted from S. aureus, particularly the EV-associated form, induces both skin barrier disruption and AD-like skin inflammation, suggesting that EV-associated α-hemolysin is a novel diagnostic and therapeutic target for the control of AD.
Introduction
Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by eczematous lesions with pruritus and xerosis [1]. AD skin lesions show distinct features, such as disrupted barrier function with epidermal hyperplasia and Staphylococcus aureus colonization. In particular, abnormal skin barrier function induced by the death of keratinocytes is one of the major causes in the etiology of AD [2,3,4]. Through the disrupted skin barrier, pathogen-associated antigens and allergens can penetrate the skin and subsequently affect host immune responses. Skin lesions of AD patients show increased keratinocyte cell death induced by immunologic mediators [5,6,7].
S. aureus is a gram-positive bacterium and notorious pathogen that can induce many human diseases [8]. Human skin and nasal anterior are well-known reservoirs of S. aureus, and it has been reported that carriage of S. aureus is related to allergic diseases. In particular, S. aureus is closely related to AD because it colonizes the skin lesions of most AD patients and augments disease severity [9,10]. S. aureus affects the host immune system by producing pathogenic molecules and toxins. For example, S. aureus produces staphylococcal enterotoxins that can induce excessive T-cell responses, as well as hemolysins, that cause cell death by forming heptameric membrane pores [8,11]. Among these, a-hemolysin (also called as a-toxin) is an important toxin, which kills many types of cells [8,12,13]. It has also been reported that a-hemolysin can target keratinocytes and is related to AD disease severity [14,15].
Recent evidence indicates that S. aureus secretes extracellular vesicles (EVs) as well as soluble toxins [16]. EVs derived from S. aureus are 20-200-nm vesicular structures that are membraneenveloped spherical complexes and contain many proteins, DNA, RNA, and toxins. S. aureus EVs show potent immunogenicity and are related to AD pathogenesis [17]. Proteome analyses showed that EVs harbor pathogenic toxins, including a-hemolysin [16]. Thus, we hypothesized that a-hemolysin, particularly the EVassociated form, is a key mediator of AD pathogenesis.
Comparison of a-hemolysin production between AD patients and healthy subjects
To evaluate the relationship between a-hemolysin production from S. aureus and AD, a-hemolysin levels were measured in culture media of S. aureus from AD patients and healthy controls. All bacteria were cultured under the same conditions and ahemolysin levels in the culture media were measured by western blot. Although two of six samples of S. aureus from healthy controls produced a-hemolysin, most S. aureus from AD patients (seven of eight) produced it (Fig. 1, A). Furthermore, when a-hemolysin production was measured in S. aureus culture media from 90 AD patients, 91% of S. aureus from AD patients were positive for ahemolysin compared to 33% of healthy controls (Fig. 1, B and Table 1). In terms of a-hemolysin production according to AD severity, a-hemolysin production was significantly higher from S. aureus from the severe group compared to S. aureus from the mild and moderate groups ( Fig. 1, C and Table 1). These findings suggest that a-hemolysin from S. aureus is related to AD disease development and/or progression.
The effect of a-hemolysin on keratinocyte cell death Numerous reports have shown that a-hemolysin can kill many types of cells, including epidermal keratinocytes [12,13,14]. Because a-hemolysin-producing S. aureus were more frequent in AD patients compared to healthy controls, we evaluated the effect of a-hemolysin on keratinocyte death by measuring cell viability after treatment with bacterial culture media. We found that HaCaT keratinocyte viability was decreased upon treatment with culture media of S. aureus from AD patients compared to healthy controls (Fig. 1, D). In addition, we evaluated the effect of S. aureus EVs on HaCaT keratinocyte death. The results indicate that keratinocyte viability was significantly decreased upon treatment with S. aureus EVs from AD patients (Fig. 1, E). These findings suggest that a-hemolysin in S. aureus EVs may be an important etiologic agent in AD pathogenesis.
The role of a-hemolysin in S. aureus EVs on keratinocyte cell death Based on these data, we sought to determine whether S. aureus EVs harbor a-hemolysin, which induces keratinocyte cell death. For these experiments, we used the S. aureus 14458 strain, a reference strain used previously [16,17]. Our data show that ahemolysin was present in both culture media and EVs from the S. aureus 14458 strain (Fig. 2, A). In terms of a-hemolysin hemolytic activity, we found that hemolysis was induced by S. aureus EVs in a dose-dependent manner, as well as by a soluble form of a- hemolysin (sHla) and the S. aureus culture media (Fig. 2, B and Fig. S1, A). Next, to evaluate the cytotoxic effect of a-hemolysin on keratinocytes, S. aureus EVs were added to HaCaT keratinocytes. Keratinocyte viability was significantly decreased upon treatment with S. aureus EVs, S. aureus culture media, and sHla compared to PBS alone (Fig. 2, C). S. aureus EVs and sHla also killed primary human keratinocytes (data not shown). To elucidate the role of a-hemolysin in EVs on keratinocyte cell death, we performed experiments using S. aureus strains that produce various amounts of a-hemolysin. We found that the amounts of ahemolysin in S. aureus EVs were positively associated with keratinocyte death (Fig. S1, B). Next, to evaluate the effect of a-hemolysin deficiency on keratinocyte death, we isolated EVs from a-hemolysin-positive WT (Newman strain) and a-hemolysindeficient mutant strains. Cell viability of HaCaT keratinocytes was significantly higher after treatment with S. aureus EVs from the ahemolysin-positive strain compared to the a-hemolysin-negative strain. In addition, keratinocyte death was reversed by treatment with EVs isolated from the a-hemolysin-negative strain that complemented a-hemolysin using a plasmid (Fig. 2, D). Furthermore, the results indicate that EVs from a-hemolysinnegative S. aureus strains from either AD patients or healthy controls did not induce HaCaT keratinocyte death, whereas death was induced by a-hemolysin-positive S. aureus strains from AD patients (Fig. 2, E). Taken together, these findings suggest that ahemolysin in S. aureus EVs are a key player in AD pathogenesis via keratinocyte death.
Comparison between soluble and EV-associated ahemolysin on keratinocyte cell death Although sHla was reported to act on the plasma membrane of keratinocytes [14], the working mechanism of S. aureus EVs on keratinocyte death remains unknown. First, we evaluated the cellular localization of EVs. When fluorescence-labeled EVs were added to HaCaT keratinocytes, EVs were internalized into the cytoplasm (Fig. 3, A). In our measurements, EVs contained approximately 0.6 mg of a-hemolysin after treatment with 10 mg of EVs (quantified by total protein amount) (Fig. S2). When HaCaT keratinocytes were treated with equal amounts of soluble and EVs forms of a-hemolysin, a-hemolysin in EVs was more effectively delivered into the keratinocytes (Fig. 3, B). Furthermore, to compare the cytotoxic effects of EV-associated a-hemolysin and sHla, HaCaT keratinocytes were treated with equal amounts of ahemolysin in the soluble and EV forms. This study showed that cytotoxicity was enhanced after treatment with EV-associated ahemolysin compared to sHla (Fig. 3, C). Keratinocyte death was induced faster by EV-associated a-hemolysin versus sHla (Fig. 3, D). Together, these findings suggest that compared to sHla, EVassociated a-hemolysin potently induces keratinocyte death.
a-hemolysin localization in S. aureus EVs
Generally, secreted soluble toxins are neutralized and lose their activity by engaging the host immune system [18,19,20]. Compared to soluble toxins, EVs may protect toxins by enveloping them with cell membrane. Our data show that EV-associated ahemolysin remained intact after proteinase K treatment; however, after EVs were disrupted by lysis buffer, EV-associated ahemolysin was degraded by proteinase K (Fig. 3, E). This finding suggests that a-hemolysin is localized in the EV lumen, not on the EV surface. Moreover, when both soluble and EV-associated ahemolysin were treated with the anti-a-hemolysin antibody, keratinocyte cell death induced by EV-associated a-hemolysin was unaffected, whereas the cytotoxicity induced by sHla was reversed ( Fig. 3, F). Collectively, these findings suggest that ahemolysin in the EV lumen enhances killing of keratinocytes and evasion of host immune defenses.
Comparison of cell death mechanisms between EVassociated and soluble a-hemolysin
Although several reports have suggested that sHla induces host cell apoptosis [12,21,22], the exact mechanism of EV-associated ahemolysin in keratinocyte death is unknown. We found that the morphology of cell death differed between soluble and EVassociated a-hemolysin; HaCaT keratinocytes were rounded and many cells were floating upon sHla treatment, which is indicative of apoptotic cell death; whereas cells underwent cell rupture upon EVs treatment, suggestive of necrosis (Fig. 4, A). In accordance with the observed cell morphology, LDH release, a marker of necrosis or cell rupture, was significantly increased after EVs treatment (Fig. 4, B). In addition, high-mobility group box (HMGB)-1, another marker for necrosis [23] was detected in culture media of EV-treated HaCaT keratinocytes, but not from that of sHla-treated cells (Fig. 4, C). Flow cytometry also showed that the number of 7-AAD-positive cells (necrotic cells) increased after EVs treatment, whereas annexin V-positive cells (apoptotic cells) increased after sHla treatment (Fig. 4, D). To sum up, these findings suggest that EV-associated a-hemolysin induces necrotic cell death by carrying a-hemolysin into the cytoplasm of keratinocytes, whereas sHla induces keratinocyte death via apoptosis.
In vivo effects of EV-associated and soluble a-hemolysin on skin barrier disruption Because keratinocytes are major constituents of the skin barrier, keratinocyte death is a key contributor of skin barrier disruption in AD pathogenesis [2,5]. To examine the in vivo effects of EVassociated a-hemolysin on skin barrier disruption, EVs from ahemolysin-positive and -negative S. aureus strains, as well as sHla, were applied to the back skin of mice. Evans blue dye penetration was enhanced by a-hemolysin-positive EVs or sHla treatment compared to PBS or a-hemolysin-negative EVs treatment (Fig. 5, A). Next, we aimed to evaluate the in vivo effects of skin barrier disruption on the penetration of high-molecular-weight allergens. Therefore, fluorescein-labeled ovalbumin (OVA) was administered to EV-and sHla-treated skin, and OVA levels were measured in excised skins by quantification of fluorescein. We found that OVAfluorescein levels were increased in mice treated with a-hemolysinpositive EVs or sHla compared to a-hemolysin-negative EVs or PBS treatment (Fig. 5, B). Together, these findings suggest that both EV-associated and soluble a-hemolysin induce skin barrier disruption via keratinocyte cell death and consequently enhance penetration of high-molecular-weight allergens.
Effects of EV-associated and soluble a-hemolysin on proinflammatory mediator production by keratinocytes Our previous reports showed that S. aureus EVs induced proinflammatory cytokine production by dermal fibroblasts and airway epithelial cells [17,24]. To evaluate the effect of EVassociated and soluble a-hemolysin on the production of proinflammatory mediators from keratinocytes, we measured the production of pro-inflammatory cytokines from HaCaT keratinocytes after treatment with equal amounts of a-hemolysin in the EV and soluble forms. We found that the cytokine production profile differed between EV-associated and soluble a-hemolysin. IL-6 was enhanced by both EV-associated and soluble a-hemolysin, IL-1b was enhanced only by EV-associated a-hemolysin, and TNF-a was enhanced only by sHla (Fig. 6, A). Moreover, IL-1b and IL-6 production by keratinocytes was decreased after treatment with EVs derived from the a-hemolysin-negative S. aureus strain compared to the a-hemolysin-positive strain, whereas TNF-a production was enhanced in the former group versus the latter group (Fig. 6, B). Collectively, these findings suggest that EVassociated a-hemolysin induces IL-1b and IL-6 production from keratinocytes but may inhibit TNF-a production induced by S. aureus EVs or other signals.
Effects of EV-associated and soluble a-hemolysin on the development of AD-like skin inflammation
Finally, we evaluated in vivo effects of EV-associated and soluble a-hemolysin on the development of skin inflammation. To do this, the same amounts of a-hemolysin in the EV-associated and soluble forms were administered epicutaneously into the mouse skin and histological alterations were evaluated. Dermal infiltration of inflammatory cells, particularly eosinophils, was enhanced by EVassociated a-hemolysin but not by sHla. However, both forms of a-hemolysin increased epidermal cell hyperplasia (Fig. 7, A). Furthermore, dermal infiltration of inflammatory cells, including eosinophils, and epidermal thickening were reduced in skin treated with repeated applications of a-hemolysin-negative EVs (Fig. 7, B). Taken together, our data show that a-hemolysin in S. aureus EVs are a key player for the development of AD phenotypes, including epidermal thickening and eosinophilic inflammation in the dermis.
Discussion
If the skin barrier is disrupted, pathogen-associated antigens and allergens can penetrate into the human body. Skin barrier disruption is considered one of the major causes of AD exacerbation, but many studies have focused on intrinsic factors, such as host molecules that maintain the skin barrier [4,25]. Though it is known that staphylococcal toxins can affect skin barrier integrity for a long time, the role of a-hemolysin in skin barrier disruption via killing keratinocytes have been reported recently [26,27]. In the present study, we elucidated the role of extrinsic factor (a-hemolysin and EVs) on the development of skin barrier disruption and AD-like inflammation. The present data show that a-hemolysin-producing S. aureus had colonized AD patients, and that soluble and EV-associated a-hemolysin induce keratinocyte cell death, consequently enhancing skin penetration of high-molecular-weight allergens. However, EV-associated ahemolysin was found to induce keratinocyte necrosis, whereas sHla induced keratinocyte apoptosis. Additionally, EV-associated a-hemolysin induced epidermal thickening and eosinophilic inflammation in the dermis, whereas the soluble form induced only epidermal thickening. This is the first report that a-hemolysin in EVs derived from S. aureus induces skin barrier disruption and AD-like skin inflammation, predominantly via keratinocyte necrosis and the production of pro-inflammatory mediators by keratinocytes.
S. aureus secretes toxins, including a-hemolysin, as both soluble and EV-associated forms. EV-associated toxins have some advantages in intercellular communication compared to soluble forms. Toxins in the EV lumen, such as EV-associated ahemolysin, can be protected from clearance by host defense systems, including antibody-mediated neutralization and proteasemediated destruction, enabling toxins to retain their function and travel long distances without interference [28]. In addition, because EVs are membrane-enveloped complexes, they can deliver their contents easily into the cytoplasm by fusing with the host cell membrane or by endocytosis after interaction between ligands on EVs and receptors on host cells [18,29]. Indeed, the present data show that S. aureus EVs were internalized into the cytoplasm of keratinocytes and that EVs efficiently delivered ahemolysin to the cytoplasm. Collectively, these findings suggest that EVs-associated toxins are key molecules in disease pathogenesis.
EVs derived from several bacteria kill host cells by transferring cytotoxic factors [29,30,31]. It was reported that S. aureus EVs can induce death of human epidermoid cancer cells [32]. Additionally, recent data indicate that a-hemolysin associated with S. aureus EVs can induce death of cervical cancer cells [33]. In addition, the present data showed that EV-associated a-hemolysin can also induce keratinocyte necrosis, whereas the soluble form induces apoptosis. This difference can be partly attributed to the differences in delivery efficacy between EV-associated and soluble a-hemolysin. We can speculate that a-hemolysin can be efficiently delivered by EVs and that a high a-hemolysin concentration in the cytoplasm attacks cell membranes and/or cytoplasm organelles, thereby initiating necrosis pathways. Cell death via necrosis or apoptosis can result in immunologically distinct consequences [22]. The present data showed that EV-associated a-hemolysin up-regulateed the production of IL-6, a key mediator of Th17 polarization, which is associated with keratinocyte necrosis. In contrast, sHla enhanced the production of TNF-a, but not of IL-6. Our previous data demonstrated that skin exposure to S. aureus EVs induced a Th17-cell response in regional lymph nodes and ultimately resulted in AD-like skin inflammation [17]. Together, these findings suggest that S. aureus EVs induce skin barrier disruption and Th17-associated inflammation in the dermis via effective delivery of a-hemolysin into keratinocytes.
The expression of toxins from S. aureus can be regulated by environmental stress, and also host factors, such as filaggrin production [11,34,35,36]. In the present study, most of S. aureus isolated from the skin of AD patients was found to produce ahemolysin, as soluble and EV-associated forms, which can induce skin barrier disruption. In contrast, we can hardly detect the production of a-hemolysin from S. aureus isolated from healthy control subjects. Moreover, the levels of a-hemolysin in lavage fluids from the AD patient skin were found to be positively correlated with AD severity. Because EVs are produced via shedding of bacterial membrane and contain many pathogenic molecules [16,18], it is hard to define the effect of EVs by deleting or adding specific molecules. We can assume that many proteins interact with other proteins and the relationship between various proteins can affect overall characteristics of EVs. Nevertheless, out present findings suggest that a-hemolysin, especially EV-associated form, is a good biomarker for the diagnosis and therapy of AD.
In summary, the present study showed that a-hemolysin, present in the EV lumen, induces skin barrier disruption and AD-like skin inflammation via keratinocyte necrosis and/or upregulation of pro-inflammatory mediator production from keratinocytes. Moreover, S. aureus colonized on AD patient skin secretes a-hemolysin, which is significantly related to AD severity. These findings indicate that a-hemolysin, particularly the EV-associated form, is a novel target for diagnosis and treatment of AD.
Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The experimental protocols were approved by the Institutional Animal Care and Use Committee at POSTECH, Pohang, Republic of Korea (Permit Number: 2011-01-0027). All animal experiments were planned in order to minimize mice suffering. The study protocol for human samples was approved by the Ethics Committee of Seoul Suncheonhyang Hospital (Permit Number: 2010-01-0016). Participants provided their written informed consent to participate in the present study.
Mice SKH-HR1 hairless mice were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan) and were bred in a specific-pathogen-free facility at Pohang University of Science and Technology (POSTECH; Pohang, Republic of Korea).
S. aureus isolation from human samples S. aureus was collected from the skin lesions of 90 AD patients visiting the Pediatric Clinic of Seoul Suncheonhyang Hospital (Seoul, Republic of Korea). S. aureus from healthy controls was isolated from the skin of the upper limbs and subungual spaces from 36 volunteers who had no AD symptoms.
Isolation of S. aureus EVs
S. aureus EVs were obtained as described previously [17]. Briefly, S. aureus was cultured in nutrient broth or tryptic soybean broth (Difco, Sparks, MD) at 37uC to an optical density (OD) of 1.5 at 600 nm. Bacteria were removed by centrifugation and filtration. The filtrate was concentrated and the resulting concentrate was filtered and ultracentrifuged at 150,0006g for 3 h. The pellet was resuspended in PBS. S. aureus-derived EVs protein concentrations were measured using BCA assays (Thermo Scientific, Rockford, IL). Hereafter, the dose of S. aureus EVs refers to the quantity of S. aureus-derived EVs proteins.
Cytotoxicity measurements
Keratinocyte viability was measured at 24 h after treatment using thiazolyl blue tetrazolium bromide (MTT) purchased from Sigma Aldrich (St. Louis, MO). The PBS control group was used as 100% viability. Lactate dehydrogenase (LDH) activity in the culture supernatant was measured using the LDH cytotoxicity detection kit purchased from Takara Bio Inc. (Otsu, Japan) according to the manufacturer's instructions.
Hemolysis measurements
Red blood cells (RBCs) were isolated from mouse whole blood. a-Hemolysin (Sigma-Aldrich, USA) and S. aureus EVs were added to RBCs and incubated at 37uC. After 1 h, the remaining RBCs were removed by centrifugation and the optical density at 540 nm of the supernatant was measured. RBC lysis buffer was used as a positive control.
Flow cytometry analyses of cell death
Annexin V (BD biosciences) and 7-AAD (Biolegend) were used detect cell death using flow cytometry. Cells were treated with S. aureus EVs or a-hemolysin, and cells in the culture supernatant and remaining cells were collected. Cells were processed according to the manufacturer's instructions. Processed cells were analyzed using FACSCalibur (Becton Dickinson, USA).
In vivo assays
For Evans blue dye assays and fluorescein-labeled OVA penetration, gauze soaked with 100-ml PBS containing S. aureus EVs or a-hemolysin was placed and secured on mildly tapestripped skin. Mice were treated five times in 1 week with S. aureus EVs and a-hemolysin. Dorsal skin was serially fixed with 30, 50, 70, and 100% methanol. After fixation, 0.1% Evans blue was added for 10 min, followed by washing with PBS. Skin was excised, immersed in formamide, and incubated at 60uC. After 6 h, the optical density at 620 nm was measured. For OVAfluorescein penetration, 50 mg of fluorescein-conjugated OVA were added twice to S. aureus EVs-or a-hemolysin-treated skin. Next, the skin was excised and homogenized. Fluorescence was measured using a Wallac 1420 Victor luminometer (American Instrument Exchange, Inc., Harverville, MA). Skin alterations were evaluated after S. aureus EVs and a-hemolysin treatment three times per week for 3 weeks, as reported previously [17].
Statistical analyses
For multiple comparisons, one-way analysis of variance (ANOVA) was used first. If significant differences were found, individual t-tests or Wilcoxon's rank-sum tests were performed between pairs of groups. Differences were considered statistically significant if P,0.05. Figure S1 A, Hemolytic activity of EVs and culture media from the ATCC14458 strain and a-hemolysin-deficient EVs strain. Hemolysis mediated by RBC lysis buffer is used as 100%. B, The amount of a-hemolysin on EVs from different strains (left panels) and cytotoxicity of EVs on keratinocytes (right panel). ** P,0.01 versus the PBS group. (EPS) Figure S2 Quantification of a-hemolysin in EVs. Western blotting showed that 0.6 mg of a-hemolysin was present in 10 mg of EVs protein from the ATCC14458 strain and 0.25 mg of ahemolysin was present in 10 mg of EVs protein from the Newman strain. (EPS) | 2017-05-20T01:03:55.652Z | 2014-07-03T00:00:00.000 | {
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6002017 | pes2o/s2orc | v3-fos-license | Multiple Effects of Bracken Fern under in vivo and in vitro Conditions
Bracken (Pteridium aquilinum) is a plant that is spread all over the world, especially in acidic forest soils, warm dark areas of both torrid, subtorrid zones, woods, and road sides (Marrs and Watt, 2006; Xu et al., 2009). Some human populations, such as Japan, Ouro Preto area in Brazil, Canada, and China, consume bracken crosiers as a nourishing and delicious food (Hirono, 1993; Chen et al., 2008; Li et al., 2008; Hojo-Souza et al., 2010). It is very nutrient-dense and a good source of protein, carbohydrates, fat, vitamins, carotenoids, and trace minerals (Li et al., 2008). In addition, bracken has numeral effects including immunomodulatory, anticancer, low blood pressure, antiviral, anti-bacterial, anti-inflammatory, and antioxidant effects. (Potter et al., 2000; Xu et al., 2009; Wang et al., 2010; Xia et al., 2010; Wang and Wu., 2013). This plant is native to the Gilan province, Mazandaran and Golestan in Iran (Khoshravesh et al., 2009; Tourchi Rodsari et al., 2012). Furthermore, the distribution of bracken-fern is noticeable in some areas such as United Kingdom (Vetter, 2009), south and Central America, South-East Asia, and Oceania (Gil da Costa et al., 2012). Unlike other ferns (Burrows and Tyrl, 2001), it does not need a lot of water and can grow in variable conditions easily. Many scientists focused their studies on this plant because of its probable effects on human health and animal. So researches began on this interesting plant mass (Wilson et al., 1998). Long-term exposure to bracken causes similar effects of ionizing radiation (Evans et al., 1968).
Introduction
Bracken (Pteridium aquilinum) is a plant that is spread all over the world, especially in acidic forest soils, warm dark areas of both torrid, subtorrid zones, woods, and road sides (Marrs and Watt, 2006;Xu et al., 2009).Some human populations, such as Japan, Ouro Preto area in Brazil, Canada, and China, consume bracken crosiers as a nourishing and delicious food (Hirono, 1993;Chen et al., 2008;Li et al., 2008;Hojo-Souza et al., 2010).It is very nutrient-dense and a good source of protein, carbohydrates, fat, vitamins, carotenoids, and trace minerals (Li et al., 2008).
In addition, bracken has numeral effects including immunomodulatory, anticancer, low blood pressure, antiviral, anti-bacterial, anti-inflammatory, and antioxidant effects.(Potter et al., 2000;Xu et al., 2009;Wang et al., 2010;Xia et al., 2010;Wang and Wu., 2013).This plant is native to the Gilan province, Mazandaran and Golestan in Iran (Khoshravesh et al., 2009;Tourchi Rodsari et al., 2012).Furthermore, the distribution of bracken-fern is noticeable in some areas such as United Kingdom (Vetter, 2009), south and Central America, South-East Asia, and Oceania (Gil da Costa et al., 2012).Unlike other ferns (Burrows and Tyrl, 2001), it does not need a lot of water and can grow in variable conditions easily.
Many scientists focused their studies on this plant because of its probable effects on human health and animal.So researches began on this interesting plant mass (Wilson et al., 1998).Long-term exposure to bracken causes similar effects of ionizing radiation (Evans et al., 1968).
Motahhareh Tourchi-Roudsari
Ion-causing compounds are related to the production of bracken radicals (Povirk et al., 2006).These are the initial effectors of DNA lesion and chromosomal aberrations.Epidemiological studies have shown association between the occurrence of neoplasms in humans and ingestion of bracken leaves (Shahin et al., 1999;Alonso-Almelot and Avendano 2001;2002), the consumption of milk from cows fed bracken fern (Alonso-Amelot et al., 1996;Cot, 2008), or the consumption of contaminated water with compounds of ferns (Rasmussen et al., 2003).
The main toxic agent in bracken is Ptaquiloside, a nor-sesquiterpene glycoside that is responsible for carcinogenic and toxic syndrome of the fern (Yamada et al., 2007).The highest concentration of PT was found in leaves and young unfolding fronds.The mutagenicity, clastogenicity, teratogenicity and carcinogenicity have been demonstrated.Some epidemiological evidences have shown higher risk of gastric and/or esophageal cancer in people who consume bracken crosiers; people who consume milk of cows that are grazing in brackeninfested areas (Yamada et al., 2007;Cot, 2008;Fletcher et al., 2011).PT has been found in milk of bracken-fed cattle through experimental tests.
Studies have revealed other toxic compounds existing in bracken fern, which depend on ptaquiloside (Castillo et al., 1998) despite having lower concentrations (Freitas et al., 2001).Furthermore, studies proved that bracken extract can cause cell death through DNA damaging by activating apoptosis mechanism.(Siman et al., 2000;Campos-da-Paz et al., 2008;Pereira et al., 2009;Tourchi Roudsari et al., 2012) It may also induce inhibition of cell growth via cell cycle arrest on cancer cells in vitro (Tourchi Roudsari et al., 2012)..
Toxicity of Bracken Fern
The inductions of toxic syndromes by ingestion of bracken fern are different in horses, cattle, and sheep (Evans et al., 1982).Ingestion of bracken fern by horses causes thiamine deficiency.In fact, each part of bracken containing thiaminase type I is able to split the thiamine molecule.The presence of heat-stable anti-thiamin factor has been reported in bracken that may be involved in the formation of a lack. in addition, the presence of thiaminase has been proved.Most features of toxicity are depressed bone marrow activity, appearance of severe leucopenia, particularly granulocyte, thrombocytopenia, the hemorrhagic syndrome, and hematuria in cattle (Perez-Alenza et al., 2006;Di Loria et al., 2012).However, toxicity is directly influenced by quantity, the time of year and the part of the plant is important too (Samecka-Cymerman et al., 2009).
Genotoxic/Cytotoxic Effects of Bracken Fern
Many studies have proved that BF extract induces genetic instabilities and DNA damage response in cells in vitro condition.Lately, researches showed that bracken extract and PT cause DNA strand breaks in gastric cells in vitro.In fact, it associates with a decrease of cell viability; induction of late apoptotic, and necrotic cells (Gomes et al., 2012).Furthermore, bracken extract induces DNA damage and its apoptosis at high concentrations.It also may cause cell cycle arrest at mild concentrations.In fact, we showed that bracken extract can have both genotoxic and cytotoxic effect in vitro (Tourchi Roudsari et al., 2012).
In agreement with our results, a previous study reported the induction of apoptosis and cytotoxicity by BF extracts in human epithelial cells through DNA damage (Pereira et al., 2009).In addition, bracken spore causes DNA strand break in human premyeloid leukemia cells in vitro (Siman et al., 2000).Other studies have shown that bracken fern extracts induce chromosomal aberrations in mammalian cells (Almeida Santos et al., 2006).Many experiments have been done on bracken fern extracts that all of them show genotoxicity/cytotoxicity of bracken in vitro (Campos-da-Paz et al., 2008;Gil da Costa et al., 2012).According to studies, scientists clearly showed that PT can be the main agent for inducing DNA damage (Pereira et al., 2009;Gomes et al., 2012).Besides, initial studies proved that ptaquiloside-DNA interactions directly lead to spontaneous deamination and DNA strand breakage (Kushida et al., 1994).However, observers showed that DNA damage occurs by different compounds in bracken extract that they are not only PT and its derivatives but also other DNA damaging compounds (Yamada et al., 2007).
Studies with evaluation of p53 gene expression have showed that DNA damage occurs during the first hours of exposure to Gastric epithelial cells.Exposure to PT leads to deregulation of some genes expression that may cause two different responses: cell cycle arrest and DNA repair (Gomes et al., 2012).
Carcinogenic Properties of Bracken Fern
Lately, results have showed that bracken extract causes cancer in mice (Gomes et al., 2012).Progression of multiple ilea adenocarcinomas was seen among rats with a diet rich in bracken fern (Evans and Mason, 1965).At the same time, occurrence of intestinal and urinary bladder tumors was induced in such rats.Then, animals were died or killed (Pamukcu and Price, 1969).Investigations showed adenomatous polyps and adenocarcinomas progress predominantly in ileum.Hirono and co-workers proposed that highest frequency of urinary bladder tumors will be developed if small amounts of carcinogens are given over long period of times (Hirono et al., 1970).In fact, many studies have been intensively done on brackenassociated bladder tumors morphologically, especially in cattle (Carvalho et al., 2006).Some studies sought a molecular approach in rats (Freitas et al., 2002) and cows (Corteggio et al., 2010;Roperto et al., 2010;Corteggio et al., 2011).
Recent studies have showed that ptaquiloside, the most imperative carcinogen agent in bracken, can induce malignant transformation in mice and supply an indepth characterization of lymphoproliferative damages.Furthermore, experiments have proved that urinary bladder is a target organ in mice as well as in other animal species (Gil da Costa et al., 2011).Observations showed that young leaves of bracken in crosier stage of growth are consumed as food by human; However, when a diet mentioned powdered young bracken fern in a ratio of 1 part by weight of bracken to 2 parts of basal diet were given to rats for 4 month, all of the rats which survived for more than 7 months from the start time of the experiment had ilea tumors.In Japan, young bracken fern is usually consumed as food by humans after its astringent taste has been eliminated according to one of the following treatments: 1) Fresh bracken fern is submerged in boiling water containing wood ash or sodium bicarbonate, and then spiced; occasionally, it is only boiled before eating.
2) Fresh bracken is soured in salt and submerged in boiling water before eating (Cheeke, 1989).The carcinogenic activity in rats that consumed a processed brackencontaining diet as a human food was investigated (Hirono et al., 1972).Tumor frequency in rats with an unprocessed bracken-containing diet was 78.5%; in contrast, in rats fed with a diet containing processed bracken treated with wood ash, sodium bicarbonate, and NaCl, tumor incidence was 25, 10, and 4.7% respectively.Findings show that the treatment markedly reduces the carcinogenic activity of bracken fern though weak carcinogenic activity was retained in bracken after treatment.
Scientists are investigating to find out the relationship between the stage of maturation of bracken and carcinogenic effects.The carcinogenicity of mature bracken was compared with young one.According to experiments, mature bracken fern had strong carcinogenic activity; however, it was weaker than immature one.(Cheeke, 1989).Furthermore, Hirono and co-workers showed development of papilomas of tongue, pharynx, DOI:http://dx.doi.org/10.7314/APJCP.2014.15.18.7505Multiple Effects of Bracken Fern under in vivo and in vitro Conditions esophagus, forestomach, and squamous cell carcinoma of pharynx in rats fed a diet containing bracken (Hirono et al., 1982).
Bracken Cancer Model
Scientists showed that DNA damage occurs at early stages of carcinogenicity, leading to oncogenic mutations.They also reported an increase in cell proliferation happening in gastric mucosa of bracken treated mice (Gomes et al., 2012).Moreover, DNA adducts and HRAS mutations are known in mammary glands of rats which receive distinctive doses of bracken dienone-a carcinogenic compound- (Shahin et al., 1998;Gomes et al., 2012).Because p53 gene usually suppresses tumors in human (Petitjean et al., 2007), researchers evaluated mutations in exons 2-4 in treated mice.The results showed the presence of BF-induced frameshift mutations in intron 2 regions (Gomes et al., 2012).The alterations in intron 2 are also able to modulate the splicing process and then influence on p53 expression (Gomes et al., 2012).In agreement, Previous studies proved p53 gene mutations induced by bracken fern (Shahin et al., 1998;Freitas et al., 2002).Researchers also suggested a model for bracken carcinogenesis (Shahin et al., 1999).Based on this model, bracken induces carcinogenesis in two distinctive levels of both cellular and molecular in vivo (Shahin et al., 1999).At molecular level, PT converts to intermediate ptaquiloside (APT) under alkaline conditions, which then goes on to alkylate DNA at N3 of adenine or N7 of guanine at 24 hours of exposure (Shahin et al., 1998).Influenced cells have the capacity to repair such damages in a short period of time; in contrast, a few of lesions lead to incorrect repairs which cause cancer.In addition, mutations in H-ras in 60% of rats were observed only after 10 weeks exposure to APT (Shahin et al., 1998).HRAS mostly activate an early event in the formation of tumors (Barbacid, 1987;Sukumar et al., 1995).Other genes that involve in this model are called p53 and neu (Gould, 1986;1995).Based on the cancer model of bracken, mutation in both of them is described at late events (Shahin et al., 1999).The neu oncogene also associates with mammary gland carcinoma in rat (Gould, 1986;1995).The tumor suppressor gene, p53 contributes several human cancers (Gomes et al., 2012).
Immunomodulatory Effects of Bracken-Fern
Recent studies revealed that ptaquiloside suppresses IFN-Y production and NK cell-mediated cytotoxicity (Latorre et al., 2009).It was also verified that selenium supplementation prevents decrease in NK cytotoxicity which is caused by p. aquilinum (Latorre et al., 2011).In 2013 researchers showed that selenium prevents carcinogenic effects caused by immunosuppressive effects of bracken fern (Latorre et al., 2013).Furthermore, ptaquiloside involved in bracken may also promote carcinogenicity by decreasing immune supervision against lately arising tumors (Gil da Costa et al., 2012).In fact, the immunosuppressive effects of bracken may help carcinogenicity indirectly due to disorder of NK cell activity (Nakahara et al., 2013).
Possible Toxic Agents in Bracken
Studies have shown that many bracken constituents have low cytotoxic effects.Therefore, they are not directly involved in acute fern poisoning.The International Agency of Research into Cancer (IARC) reported that, there is enough evidence for carcinogenicity of bracken in animals (IARC, 1987).Although in human, the results are the same but insufficient for proving (IRAC, 1985).Chemical constituents of bracken are very varied and they depend on habitat (Chen et al., 2008).
Ptaquiloside is a potent toxin found in ferns.It was first isolated in 1983.In fact, the detection of ptaquiloside was a landmark in bracken research (Yamada et al., 2007).Fortunately, Methods for quantifying ptaquiloside in bracken have been developed (Bonadies et al., 2004;Jensen et al., 2008;Bonadies et al., 2011).Unlike rats treated with low PT content, Bracken fern containing high content of PT leads to many tumors in ileum and urinary bladder among treated rats.(Smith et al., 1988).PT has been known as a strong carcinogen.In addition, it was described to be responsible for the biological effects of bracken, such as acute bracken poisoning, bright blindness in sheep, mutagenicity, clastogenic effects and genotoxicity (Yamada et al., 2007).Recently, intraperitoneal ptaquiloside administration to CD-1 mice resulted in high level of urinary bladder dysplasia and a B-cell lymphoproliferative malignancy (Gill da Costa et al., 2011).PT should be considered highly as a carcinogenic compound for human.PT can be easily transmitted from milk to human; therefore, it is necessary to be cautious about cattle grazing (Wilson et al., 1998).Hirono's group also showed that using PT via oral route induces thrombocytopenia, myeloid aplasia, and neutropenia in a calf typical of the acute haemorrhagic syndrome (Hirono et al., 1984).
Cyanogenic glycosides are enzymatically hydrolysed by B-glucosidase, thereby hydrocyanic acid (HCN) and glucose are released.(Gil da Costa et al., 2012).All bracken varieties are not cyanogenic and prunasin is the only reported one which is thought to act as a feeding deterrent against insects and herbivore mammals, due to its acrid taste.(Alonso-Amelot and Oliveros-Bastidas, 2005).Prunasin is abundant in young crosiers (Oliveros-Bastidas and Alonso-Amelot, 2010).
Shikimic acid has not shown many evidences for carcinogenicity in animals studies and there is not sufficient evidence for Its carcinogenicity in human.Although it is unlikely to be a carcinogenic starting agent, it might act as a carcinogenic stimulating factor in bracken fern (Jones et al., 1983).
Thiaminase is a compound found in all parts of bracken.It leads to destruction of thiamin and subsequent health problems like beriberi in animals fed bracken fern.Plant Thiamine reaches to its maximum amount in summer which will remain even after plant withered state.After bracken collection and storage, thiamin is remained and can increase toxic effects (Barr and Reagor, 2001).
Quercetin is dose-dependent in vitro (Rahman et al., 1992), but not in vivo (Utesch et al., 2008).Although some scientists suggested that quercetin-induced mutations might contribute to the appearance of a malignant phenotype in vivo (Borzacchiello and Roperto, 2008), it is not seem to play a role in carcinogenicity of bracken fern ( Van der Hoeven, 1985).Indeed, Qu has pro-apoptotic effect that leads to cell damage and cytotoxicity.These effects of Qu may be resulted from various pathways (Gibellini et al., 2011).For example, the ability of Qu to induce apoptosis through mitochondrial pathway has been confirmed in U937 cell line (Ferraresi et al., 2005;Lugli et al., 2005).Even antiproliferative effects of Qu could be related to the ability of Qu for binding to tubulin which stimulates cellular microtubules depolymerization (Gupta and Panda, 2002).Furthermore, studies show that Qu as a pro-apoptotic flavonoid acts specifically on tumor cell lines rather than normal ones.The studies show that Qu is eliminated due to limitation of absorption completely with metabolism in the intestinal, decomposition completely, and its toxic effects (Staviric, 1994).So, it seems that Qu exists only in vitro.
Freshly, there was a study about constituents of bracken grown up in Yannan of China.5-Hydroxypyrrolidin-2-one, Shikimic acid and glycerol 1-stearate were extracted from young fronds of the plant for the first time (Chen et al., 2008).
Chemistry of PT Reactions
PT is unstable under acidic and alkaline conditions.It is rapidly hydrolyzed with water and produces Ptaqilosin with the lack of sugar units.It rapidly decomposes to Pterosin B in acidic conditions (Yoshihira et al., 1971;Fukuoka et al., 1978;Gomes et al., 2012) or it is converted to an unstable intermediate compound in a weak alkaline solution.This compound can activate a carcinogen called Dienone (APT) that acts as a powerful alkylating agent (Ojika et al., 1987).Dienone alkylate purin bases of DNA are naturally removed from DNA as alkylated parts.Then breaks occur in DNA structure and carcinogenicity begins (Radostits et al., 2000;Dawra and Sharma, 2001).(Figure 1)
Bracken Fern Problems in Livestock
During the periods of reduced food availability, domestic animals (herbivores and pigs) eat bracken easily and willingly, especially its tender young parts (shahin et al., 1999).The effects of bracken ingestion are diverse, depending on the animal species and the ingested dose(s) (Vetter, 2010).
Acute toxicity with bracken
It rarely occurs and there is low fatality.However, in some cases high numbers of incidence and mortality are observed (Radostits et al., 2000).Estimation the amount of plant toxicity is different in cows fed bracken.During 1 month, they probably show clinical signs and die (Humphreys, 1988).
Acute hemorrhagic syndrome (loss of blood)
Ptaquiloside is the major toxin which is responsible for bone marrow suppression and carcinogenic activities observed in ruminants.It is a lactone molecule discovered in highest concentrations in young growing parts of the plant (Perez-Alenza et al., 2006;Di Loria et al., 2012;Panter et al., 2011).
The acute hemorrhage and sudden death occur as a result of bone-marrow depression of subsequent thrombocytopenia (the condition of having a low platelet count) and increment trend to bleed (Samecka-Cymerman et al., 2009).
Enzootic Hematuria in cattle
It is a chronic and serious disease in cattle (Perez-Alenza et al., 2006;Di Loria et al., 2012).Consuming 1-2 kg of bracken for 10-15 months by cows leads to Hematuria (Van Metre and Divers, 2002;Panter et al., 2007;Lucena et al., 2011).A recent report indicates the occurrence of a similar syndrome in buffaloes and sheep (Somvanshi, 2011;Dawra and Sharma, 2001;Radostits et al., 2000).Prevalence of disease is various in different regions.This disease occurs in both sexes.However mortality is higher in male animals due to obstruction of urinary tract by blood clots (Dawra and Sharma, 2001).The hematuria forms varied tumors, especially hemangioma in the bladder wall.PT in bracken has been known as a toxic compound that causes bovine enzootic haematuria.It also induces bladder tumours in laboratory animals (Hirono et al., 1987;Radostits et al., 2000;Dawra and Sharma, 2001).The oncogenesis of PTinduced bladder has not been investigated completely, but a number of experiments have demonstrated its molecular and morphological features (Carvalho et al., 2007;Guidi et al., 2008;Corteggio et al., 2010;2011;Pires et al., 2010;Roperto et al., 2010).
Blindness in sheep
Long-term consumption of bracken by sheeps leads to the formation of nonfatal diseases such as "Progressive retinal degeneration" or bright blindness (PRD).(Somvanshi and Ravisankar, 2004;Perez-Alenza et al., 2006;Di Loria et al., 2012)
Neoplasia in gastrointestinal tract
Neoplasia in gastrointestinal tract including jaw and liver is observed in adult sheep feeding on bracken fern.The reports show that there is no relationship between tumor formation and the presence of HPV.There is also high incidence of colon and bladder tumors in cattle fed in lands covered with bracken (Angus and Sharman, 1991;Radostits et al., 2000).
Food
Fern leaves have edible consumption in Japan, Brazil and Canada (Hirono, 1993;Hojo-Souza et al., 2010).Studies have revealed the dependence between bracken consumption and the stomach and esophagus cancer in human (Wilson et al., 1998).Daily intake of bracken in japan increases the risk of oesophageal cancer about 2.1fold in men and 3.7-fold in women (Kamon and Hirayama, 1975;Hirayama, 1979).Possible risk of consumption of animal meat that fed bracken doesn't have been studied and is still unknown (Wilson et al., 1998).
Milk
Risk of digestive tract cancer exposed to bracken has been studied in South and Central America (Villalobos-Salazar, 1985;Alonso-Amelot and Avendano, 2001).Carcinogenic compounds have been secreted from milk and urine of livestock that consume bracken fern.Moreover, it is proved that it can cause cancer in laboratory animals.The studies show that even the activity of carcinogenic compounds in the milk of animals fed on bracken is more than bracken compounds (Alonso-Amelot et al., 1996).Recently, Ptaquiloside has been detected in meat and milk from cattle consuming bracken (Bonadies et al., 2010;Fletcher et al., 2011).Pasteurisation was found to decrease ptaquiloside concentrations in milk by 50% and a 75% decrease by boiling (Villalobos-Salazar et al., 2000).Therefore, boiling the milk before drinking it may be a helpful recommendation for people who live in rural areas suspected of being exposed to considerable amounts of Ptaquiloside (Gil da Costa et al., 2012)
Water
Water contamination may also occur.Ptaquiloside leaching from fronds exposed to rain may subsequently contaminate soil and groundwater (Ayala-Luis et al., 2006;Engel et al., 2007;Ovesen et al., 2008;Jensen et al., 2008).There is no evidence to prove that water contamination with bracken fern can cause disease in human; nonetheless, a number of toxic compounds in fern are soluble in water and probably can be entered into water tanks (Evans et al., 1984).
Inhalation and ingestion of spores
Spores are proved to be one of the ways of ptaquiloside transfer from bracken to humans (Rasmussen et al., 2013).Therefore, this finding is very probable to describe the toxicity of bracken spores for laboratory mice and human cell lines (Rasmussen et al., 2013).Lymphocytic leukaemia has been reported in mice fed bracken spores, and bracken spores have proven to have genotoxic effects for human cell lines, including gastric cells (Siman et al., 2000;Gomes et al., 2012).The fern spores have been identified as a carcinogenic agent in mice (Evans, 1979).Inhalation and ingestion of spores can be carcinogenic in humans (Wilson et al., 1998).The study is only about direct ingestion of spores not the effects of animal exposure to airborne spores (Wilson et al., 1998).The research showed that, spores administered by gavage produce DNA adducts in mice (Povey et al., 1996).The significance of these results requires further examinations.
Skin
There is no finding about the effects of ferns on humans and animals via direct contact with skin; however, this pathway would seem an unlikely cause of disease (Wilson et al., 1998).
Studies on Human Health
Most speculations about harmful effects of bracken on human health is based on its effects on animals, but it is not possible to reach a conclusion for human based on experiments on model animals; However, researchers conclude that there is a direct connection between bracken consumption and stomach cancer in men and women and esophageal cancer in men only (Wilson et al., 1998).The risk of esophageal cancer that associates with the consumption of bracken is estimated to be about 2.1-fold in men and 3.7-fold in women (Kamon and Hirayama, 1975).The highest incidence of stomach cancer was found in those who have spent their childhood in covered lands such as Gwynedd region (Galpin et al., 1986;Galpin et al., 1990).The fact that consumption of bracken and stomach and esophagus cancer are not related to each other was known substantially in Brazil (Marliere et al., 1995).The association between ferns and cancer in has been investigated in England and Wales (Wells and McNally, 1989).Villalobos-Salazar and co-workers compared incidence rates of stomach cancer among people who were born in bracken infested and bracken free areas in Costa Rica considering their age (Campo et al., 1990).Brackeninfested and bracken-free were determined according to height above sea level, which is a confirmed method in previous studies.It was reported that the number of gastric and esophageal cancer victims among women and all people who were born in bracken infested areas were at least twice than those who were born in bracken free areas (Wilson et al., 1998).
High daily consumptions of pickles, edible wild plants, especially bracken crosiers, were significantly related to increase of pancreatic cancer risk, particularly in men, who had a relative risk of 2.98 (95%CI=1.46-6.07)(Ramwell et al., 2010).
In a study of 783 patients with stomach cancer and 1566 hospital controls in Hiroshima and Miyagi, a higher risk of stomach cancer was known in Japanese farmers and totally among Japanese population, not other jobs or even Hawaiian-Japanese (Haenszel et al., 1976).An association was known between bracken consumption and stomach cancer.Japanese higher consumption rates of Bracken accounts for mentioned population differences (Ramwell et al., 2010).The association between dietary intake of bracken and esophagus cancer was investigated in 98 cancer patients and 476 control cases aged 60 or older in Japan.
Control of Bracken
According to the plant's rhizomatous rooting system and its ability for producing a large amount of spores, spread and control of bracken fern can be difficult (Bebbington and Wright, 2009).Growth can be controlled by regular cutting of mature plant or by deep plowing over time (Burrows and Tyrl., 2001).Moreover, herbicide may be an effective control if teamed with close cutting to encourage vegetative growth before a treatment is applied (Burrows and Tyrl, 2001).
Conclusion
The toxicity of bracken fern and its constituents, mainly ptaquiloside, have been investigated in studies of farm and laboratory animals and in human epidemiological studies.Bracken fern, a widespread plant, is toxic for many animal species.It can affect a high percentage of animals and many body systems.The observations show that most of the people who work or play among bracken ferns, are at risk for cancer, particularly in spring; besides, people who eat bracken as vegetable or drink milk from cows that are fed bracken are endangered.Bracken fern extract is a potent source of anticancer compounds.It can be utilized pharmaceutically.Further studies, are required to clarify the biological relevance of this response. | 2017-09-07T03:07:56.445Z | 2014-10-11T00:00:00.000 | {
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225444620 | pes2o/s2orc | v3-fos-license | Use of Humidified High Flow Nasal Oxygen in Community Palliative Care: A Case Report
Breathlessness is a distressing symptom that is often seen in palliative care patients with respiratory failure and it can make care in the home setting difficult. Humidified High Flow Nasal Oxygen is a relatively new intervention for respiratory failure, but it has not been researched greatly in a palliative care setting. One device with the capacity to deliver high flow humidified oxygen to spontaneously breathing patients is the myAIRVO2 humidifier.1 The myAIRVO2 is a humidifier with an integrated flow generator that delivers warmed and humidified respiratory gases to a spontaneously breathing patient.1 The following case report describes how the technology was used at home for symptom control in a 76 year old patient with severe chronic obstructive pulmonary disease with associated pulmonary hypertension. The patient was successfully discharged from hospital and managed at home using high-flow nasal oxygen for approximately one month up until his death. In this last month of life, he reported that he was more comfortable on high-flow nasal oxygen than on traditionally-administered oxygen. Humidified High Flow Nasal Oxygen is potentially beneficial to aid in symptom control for palliative care patients in an inpatient and community setting.
Introduction
Humidified High-Flow Nasal Oxygen (HHFNOx) has been gaining in popularity and has become an important treatment in critical care in recent years. 2 This technology is now starting to be trialed in both palliative care inpatient and community settings. One device with the capacity to deliver high flow humidified oxygen to spontaneously breathing patients is the myAIRVO2 humidifier with integrated flow generator. This system works with an air-oxygen blender allowing for a fraction of inspired oxygen (FiO 2 ) of between 21% and 100%. It can generate high flows of air up to 60 L/min. 3 The use of HHFNOx has a range of physiological benefits including pharyngeal dead space washout, reduction of nasopharyngeal re-sistance, positive expiratory pressure effect, alveolar recruitment, humidification, and reduction in discomfort of dyspnea. 3 Although there is an increasing amount of experience on the use of HHFNOx in healthcare, there is very limited research on its use in a community palliative care setting.
Routine palliative care management of dyspnea involves a multidisciplinary team approach. After investigating and treating reversible causes of breathlessness, various symptomatic management strategies can be useful in improving quality of life. Patients may be provided with a breathlessness action plan and receive education on various coping strategies including the use of fans, modifying positioning for comfort, breathing exercises, and energy conservation techniques.
Various medications can also relieve symptoms, including opioids and benzodiazepines. 4 Supplemental continuous oxygen is sometimes prescribed for patients with dyspnea, primarily for those with documented hypoxemia. 5 Patients with end stage respiratory failure, such as those with severe chronic obstructive pulmonary disease (COPD) often develop increasing oxygen requirements, ultimately requiring continuous home oxygen 24-hours a day. For patients on higher oxygen flow rates, for example, on 10-15 L/min through face mask, it can be very difficult to remove the face mask for a few minutes for everyday tasks such as eating and drinking because of severe dyspnea.
One clinical benefit of HHFNOx it that patients are typically delivered warmed high flow of oxygen through nasal cannula and this is better tolerated than traditional non-warmed oxygen. 6 Also, because high flow rates of oxygen are delivered through nasal cannula rather than through face mask, patients are able to speak, eat and drink more easily. HHFNOx with its higher flow can improve dyspnea to a greater extent than traditional oxygen as it decreases work of breathing. 7 When patients are discharged home from hospital they usually need to be weaned from HHFNOx back onto oxygen through traditional nasal cannula or face mask. This is especially true in districts where HHFNOx machines are not available in the community. Having an option to trial HHFNOx in the community can help facilitate home discharge for patients who are too unwell to be weaned permanently from the HHFNOx in hospital. This could potentially reduce the length of stay for some patients and allow patients to be cared for at the site of their choosing. It may help facilitate dying in the home setting rather than the hospital setting if this is in line with the patient's wishes. Using HHFNOx in the community setting may also help patients stay at home longer as there is evidence that humidification can reduce acute exacerbations of airways disease and increase time until the first exacerbation. 8 Our case report describes one patient who trialed HHFNOx in a community setting. Written informed consent was obtained from the patient's wife after the patient died.
Case Description
A 76 year old man with severe COPD with associated pulmonary hypertension was admitted under the respiratory physicians at the Gold Coast University Hospital after presenting with a non-productive cough and shortness of breath. The patient was referred by his re-spiratory physician to the palliative care team for consultation asking for advice on ongoing management of his dyspnea in the community. From the patient's perspective, shortness of breath was the main symptom concerning him. Before admission he had been on continuous home oxygen at 6 L/min.The oxygen was delivered through two oxygen concentrators run in series. He did have portable oxygen bottles but no longer used them as he became house bound before his admission. His recent usual oxygen saturations (SaO 2 ) were between 75 and 85%. He lived with his wife and had been essentially house bound for several months. He was able to mobilize short distances limited by his breathlessness. He had not been receiving any additional community nursing services; his wife had been assisting him with all his care needs.
His medication list was as follows: Seretide 250/ 25 mcg two puffs twice daily; tiotropium daily; clopidogrel 75 mg daily; aspirin 100 mg daily; bisoprolol 1.25 mg daily; furosemide 40 mg daily; oxycodone immediate release 5 mg two-hourly as required and atorvastatin 80 mg daily He was reviewed by the hospital's consulting palliative care physician who recommended low dose immediate-release oral morphine as required for dyspnea and who also arranged for the patient to be linked with the Gold Coast's community palliative care team once he was discharged.
During the patient's hospital stay an MET (Medical Emergency Team) call occurred due to an episode where his SaO 2 dropped to around 60%. This occurred after the patient had a shower and walked back to his bedside chair. He became severely breathless and anxious with an increased work of breathing. During the MET call, the patient initially responded well to increased oxygen on a non-rebreather 15 L/min mask after 20 minutes. He was then placed on HHFNOx at 50 L/min with an FiO 2 of 50%. The respiratory team's goal over the following days was to wean the patient off HHFNOx, aiming for SaO 2 to range between 70 and 90%. The patient used his medications as per the abfovrementioned list during this time.
For the following next few days, the patient was unable to be weaned from HHFNOx successfully as he developed increasing and intolerable breathlessness each time an attempt was made to switch the mode of oxygen delivery to a face mask. Escalating doses of immediate release opioids were unable to alleviate his symptoms adequately and side-effects precluded further uptitration. His respiratory team also felt benzodiazepines were contraindicated in the patient. The respiratory and palliative care teams discussed further options and made a decision with the patient to trial HHFNOx in the community setting as his goal was to return home. The palliative care and respiratory physiotherapists educated the patient and his wife on setup and safe use on HHFNOx.
Shortly after discharge, the patient was reviewed at home by the palliative care physiotherapist. On assessment the patient's oxygen saturation was 78% on myAIRVO2 with a flow rate of 40 L/min, FiO 2 of 36% and the temperature set to 35°C. The patient reported that he was using HHFNOx almost continuously but that he would replace it with a Hudson face mask with 8 L/min of oxygen when he needed to mobilize to the bathroom. He reported that he felt much more comfortable when he used the HHFNOx when compared with the face mask. Although the patient did not complete any formal quality of life outcome measures he did report that he was very happy to be at home with his wife. He also reported that he particularly enjoyed that he could drink cups of tea and have good conversations with his wife when using the HHFNOx.
The patient remained at home for eight days after his discharge. Unfortunately his HHFNOx machine needed to have a filter change. As the community palliative care staff were unavailable to troubleshoot how to replace the filter for a weekend the patient needed to return to hospital as his breathlessness and anxiety increased. The next day the patient was discharged home with the HHFNOx machine having had its filter change. The patient remained at home for three weeks during which time he slowly deteriorated in terms of function. He died at home and his wife reported that he was comfortable up until his death. His wife's only negative comment about the HHFNOx was that it required daily changes of the water chamber.
Discussion
This case demonstrates that HHFNOx can be used in a community palliative care setting to improve symptoms and quality of life and allow a patient to be cared for in the site of their choosing. In our case, HHFNOx allowed our patient to return home rather than remain in hospital for his end of life care. If HHFNOx was not available for this particular case, then the patient would have been required to remain in hospital as his symptoms were inadequately controlled to allow discharge without HHFNOx.
This case also illustrates some logistical issues with HHFNOx at home. In particular, maintenance of the machine is of key consideration. As there was no available staff member to review the machine during the weekend, our patient was required to return to hospital. Other logistical issues obviously include the availability of AIRVO or alternative HHFNOx devices in the community and the cost of purchasing or hiring these devices.
It is uncertain if the use of HHFNOx prolonged the patient's life. Without HHFNOx the severe symptoms may have necessitated higher doses of opioids and the addition of benzodiazepines and so HHFNOx did possibly allow for a longer life with less drowsiness that would otherwise have been possible. From an alternative perspective, it is possible HHFNOx prolonged the dying process for our patient. However, continued use of HHFNOx was consistent with two of the patient's goals, namely, reduced breathlessness and returning home for end-of-life care. Before commencing a trial of HHFNOx in a palliative patient it would be reasonable to require an assessment of patient function and alertness first. For example, breathless patients who are alert and still able to eat and drink may have some form of improvement in quality of life with the device. Conversely, patients who are drowsy and no longer able to eat and drink are unlikely to benefit from HHFNOx and should not be offered this therapy. There is a need for further investigation of HHFNOx in a palliative care setting to determine when it is likely to be of benefit and improve quality of life.
In our patient's case, formal outcome measures such as dyspnea scores and quality of life measures were not completed. In the future some quantitative research on the use of HHFNOx in an inpatient and community palliative care setting would be beneficial to more accurately ascertain benefits of HHFNOx for patients. | 2020-09-10T10:15:56.280Z | 2020-09-03T00:00:00.000 | {
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236601185 | pes2o/s2orc | v3-fos-license | Load Disturbance Conditions for Current Error Feedback and Past Error Feedforward State-Feedback Iterative Learning Control
Iterative learning control is a controlling tool developed to overcome periodic disturbances acting on repetitive systems. State-feedback ILC controller was designed based on the use of the small gain theorem. Stability conditions were reported in the case of past error and current error feedback schemes based on Singular values. Disturbances acting on the load of the system were reported for the case of past error feedforward only which kept the investigation of the current error feedback as an open question. This paper develops a comparison between the past error feedforward and current error feedback schemes disturbance conditions in singular values. As a result, the conditions found highly support the use of the past error over the current error feedback.
Introduction
Robot manipulators can be provided to undertake a pick and place operation with repeated executions of a finite duration task. In gantry robot application, an object is collected from a fixed location point, transferred with predefined period to be placed at another specified location. Once job is complete, the robot returns to the starting location point to repeat the same task repeatedly. Thus, the objective is to follow a prescribed reference as closely as possible for as many times as possible before the need for resetting. Other applications arise having the same operation manner, such as chemical batch processes, petrochemical processes, and microelectronics manufacturing [1].
In this paper, a trial is the known terminology for each completed job, and the trial length is known to define the finite duration. The reference trajectory with finite time duration is denoted by ( ) r t over the period 0 t T ≤ ≤ < ∞ , where T denotes the trial length. After each completed trial, information generated is available to compute the control input to be applied to the next trial. Let the integer . Iterative learning control (ILC) is a developed controlling tool that had been especially devoted to systems operating in the mode described earlier [2]. The basic principle behind it is to construct a set of control inputs { } k u such that the error sequence { } k e converges to zero in k trial after trial. In industrial applications, it is acceptable to design the controller such that the error convergence to within a specified tolerance.
For any instance on the current trial, information from any instance on the complete previous trial can be used assuming all previous trial data is available; noncausal temporal information.
Several works reported the development of ILC control designs [3] [4]. Those introduced the development of the ILC principle starting from the early reported work in [2], where a D-type ILC law was introduced having the following form , where 0 λ > is the phase-lead term and the system sampled with p denoting a sampling instant along a trial. This track of course faces cases where such control laws are insufficient to achieve the required control performance (or not capable of controlling the dynamics) and this has led to the development of model-based designs. [4] is a good starting point for the literature.
In this paper, we produce the missing disturbances condition for the current error feedback state ILC based on the framework reported in [5] and [6]. The framework assumes periodic disturbances acting on system input. The framework designed controller explicitly incorporates current error feedback, whereas the work of [7] presented a modified framework that incorporated past and current error feedback in designing the ILC controller. The idea behind the two designs depends on the internal model principle [8] with isolating the delay model and introducing a controller that stabilize the overall system around the delay operator. In both designs, past and current error cases, the disturbances acting on system input are not considered.
There are several studies that reported the uncertainty or disturbances issues such as those in [9] [10] [11], where either a robust adaptive control design was investigated for a class of nontriangular nonlinear systems in the case of unmo-deled dynamics and stochastic disturbances or for an adaptive fuzzy tracking control design problem, as well, for uncertain nonstrict feedback nonlinear SISO systems. [11] investigated the load disturbances for state feedback control in the duality framework for past error feedforward, whereas [7] did not consider such a problem within the framework that might affect the quality of the response obtained. This paper, extends the disturbance conditions in ILC design for the current error feedback case alongside the previously reported case, which concerns the past error feedforward case. A comparison is made on the results obtained for the new results which was left for the reader to dig for in [11].
The next section gives a brief background on the work of [7]. Then, the new results for past error feedforward and current error feedback in load disturbance presence are given. Finally, the overall conclusion and possible future investigation are clarified.
Background
Here we revise the ILC design introduced in [7] starting with considering a linear MIMO system S of m outputs, p inputs, and n states. The following space describes the overall transfer function in the state space form in discrete linear time-invariant, where the matrices f, Ξ, C, and D are of dimensions that keep the previous equation valid.
The system output y(z) of size m × o and u(z) is the system input of size p × o, then the output y(z) can be represented in the form of y(z) = S(z)u(z). It is known that, in ILC, the system operates a single trial of fixed time, then it resets to its original position waiting to start the next trial. Thus, a single trial with finite duration can be considered to express system dynamics over a single trial to be ( ) where 0 1 i N ≤ ≤ − with N defined as the number of trial samples. With no loss of generality, it is acceptable to consider the initial value x 0 = 0 due to resetting condition. The form presented in Equation (1) can be defined in 2 dimensions propagation. This is reflected on the start of the ILC Field for both continuous time domain and discrete field, where the latter forms the natural base to the ILC interest due to its nature of storing data. Numerous ILC designs recently rely on lifting the discrete expression to be one notational form, the index trial notation, see [4]. The lifted expression starts with introducing the input and output supervectors; u and y in iteration index A feedback connection is used to stabilize the system along the trial, where sys- The reference ( ) r t is also defined to hold the vector form in discrete space as Through measuring the error, the ILC objective is to use the measured error as a forcing function added to previous trial input to produce next trial input signal such that the system output follows the reference trajectory accurately as the trial index tends to infinity.
[12] defined a periodic signal of length N in the discrete-time formation as where the N × N matrix F w is given by The objective is to design the controller ( ) K z such that, the overall closed-loop system is asymptotically stable, the tracking error k k e r y = − tends to zero along the trial domain, and the two previous conditions are robust.
[7] extended the work presented by [5] [6] to design the ILC controller in several design schemes. The first was with state feedback
( )
G z is the overall transfer function of the system and ( ) S z is the plant model [5]. For the past error feedforward case, the stability condition is For the current error feedback, the stability condition is where G(z) in both cases is governed by the following The design presented in [7] did not consider the case where disturbance might act on the system load. [11] considered only the case with past error feedforward. Here in this paper, we investigate the case of current error feedback and compare the condition found to that found in [11].
Load Disturbance Limitation in State-Feedback ILC
As a start, [11] defined the system described in (1) where k represents the iteration index and q is being the forward shift operator. The time delay operator, δ , is inserted in the output equation. Without loss of generality, it is assumed that the process matrix ( ) S q has no delay. In the beginning of each trial, the state of the system is assumed to start from a fixed position. The number of samples in one iteration is N δ + . Now, if a control action takes place at time 0 t = , the system will respond when t δ = . Thus, it is trivial to control the output The load disturbance vector k d is analogous to ( ) k u t ; and the measurement disturbance k n , the measured output vector k y , and the reference vector r are defined analogous to (8). Required assumptions are made about k d and k n where 1) their mean is zero, weakly stationary random variables with bounded variance; 2) they are uncorrelated with each other; and 3) they are uncorrelated between iterations. Examining load disturbance limitation to assure system performance, let us start with the stability condition described in (5) for the state feedback design with past error feedforward case as well as the output described in (7) to form the following path using the singular values as a more restrictive region: which can be further reformed to the following: This can be directed to isolate the load disturbance in one hand after some manipulation and maximize its effect to form the following equation: This condition clearly says that the maximum singular value of the load disturbance acting on the present trial has to be less than the maximum singular value of the difference of the sum of all previous trials output eigenvalues minus the sum of previous trial load disturbances and the initial input response as well as the minimum singular value to the last trial control action. This makes the range where the load disturbance acting on any trial k is very restrictive and has a small range of variation in terms of its maximum singular value. The second part of the right-hand side of (9) can also be modified to give the form of ( ) ( ) ( ) , which makes the condition given in (9) as In the case of current error feedback, the load disturbance limitation condition is derived the same. The starting point is again the stability condition given in (6). The load disturbance can happen at any time instance in trial k and it is not periodic. Thus, it must be in a form that contains its weight of direction such that its effect can be analysed and suppressed. This is again considered through singular value analysis, where the maximum singular value representing the disturbance must be contained in stability region.
And this can be rewritten as As it can be seen, the resulting condition (12) states that the maximum singular value of the disturbances has to be greater than the sum of all trials output singular values but not to include minimum singular values of sum of past output signals, past disturbances, initial output as well as the maximum singular value of past disturbances and initial output, also not exceeds a value of 1. This is hard to achieve in the presence of an easier method such the past error feedforward with better feasible region of disturbance suppression.
Overall, the result found in current error feedback (12), does not support the use of current error feedback against the presence of easier case. Also, it clearly points to the advantage of using the past error feedforward controller due to its simple structure and the ease of the applying the load disturbance limitation conditions in comparison of the current error feedback case in ILC state feedback design.
Conclusion
Two ILC state feedback schemes have been investigated based on load disturbance conditions, past error feedforward and current error feedback. The results obtained in (10) and in (12) show the advantage of using the past error feedforward over current error feedback in term of the region of disturbance suppression found. The past error feedforward scheme has wider region of disturbance limitation condition compared to the current error feedback case, where it has less region of disturbance limitation. In the future, experimental verification would be valuable to support the results found and investigation of the output injection scheme is still an open area to find its conditions of stability in term of load disturbance presence.
Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper. | 2021-08-02T00:05:22.400Z | 2021-05-17T00:00:00.000 | {
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257507110 | pes2o/s2orc | v3-fos-license | Sex-specific effects of a parasite on stress-induced freezing behavior in a natural beetle-nematode system
Some animals react to predation threats or other stressors by adopting a freezing posture in an attempt to avoid detection, and the duration of this behavior usually corresponds with individual personality, such that timid individuals freeze longer. Despite decades of research on this or related behaviors (thanatosis), never has the impact of parasitism been considered. Parasites could prolong the duration, if hosts are less motivated to move (i.e. lethargic), or they could reduce it, if hosts are motivated to forage more to compensate for energy drain. We examined this behavior within a natural beetle-nematode system, where hosts (horned passalus beetles, Odontotaenius disjunctus) are parasitized by a nematode, Chondronema passali. We exposed beetles (n = 238) to four stressors in our lab, including noise, vibration, light and inversion, and recorded how long they adopt a frozen stance. Afterward, we determined nematode burdens, which can range from dozens to hundreds of worms. Beetles tended to freeze for 20 seconds on average, with some variation between stressors. We detected no effect of beetle mass on the duration of freezing, and this behavior did not differ in beetles collected during the breeding or non-breeding season. There was a surprising sex-based difference in the impact of nematodes; unparasitized females remained frozen twice as long as unparasitized males, but for beetles with heavy nematode burdens, the opposite was true. From this we infer that heavily parasitized females are more bold, while males with heavy burdens would be more timid. The explanation for this finding remains elusive, though we can rule out many possibilities based on prior work on this host-parasite system.
Introduction
Across the animal kingdom, different species have evolved a wide variety of anti-predator strategies that fit their lifestyle, body design and behavior. When directly faced with a predator or perceived threat, some animals react by feigning death as an attempt to fool the predator [1][2][3][4]. In some scientific disciplines this is referred to as thanatosis [5,6] or tonic immobility [7,8]. There can be many forms of this behavior, such as curling the abdomen a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Importantly, one recent study also demonstrated that nematode-parasitized beetles appear to break down more wood during their lives [17], which could be construed as evidence that they are more motivated to eat, perhaps as a compensatory mechanism against the energy drain of the nematodes. Given this finding, an interesting follow-up question relates to the host behavior when dealing with perceived threats. If indeed the parasitized beetles are simply "hungrier", does this mean they would be less motivated to adopt any anti-predator behavior that necessitates inactivity? Another follow-up question would be to evaluate if the impact of nematodes on female behavior differs from males; prior work with this system also indicated that female beetles are inherently more motivated to be active than males [34]. Here, we describe a series of experiments using this beetle-nematode system that were designed to address these questions, with results yielding a surprising answer.
Methods
We report results from two identical experiments, which, importantly, were conducted in different phases of the horned passalus beetle life cycle. The first experiment was completed during summer (June-July) 2021, which overlaps with the beetle breeding and grub-rearing phase [32,33]. Here we tested a total of 140 beetles. Our second experiment was conducted during Jan-March 2022, which corresponds with the beetle wintering phase; by this time, all young from the prior summer and fall have completed metamorphosis, and adult beetles are mostly dormant in logs (author, pers. obs.). At this stage they are not in any form of physiological diapause, and when brought to the lab and placed in room temperature, they appear to resume normal activity and feeding (pers. obs.). In this experiment we tested 98 total beetles. All collection and laboratory procedures were the same in each experiment.
Beetle collection and housing
For each experiment, beetles were hand-collected from nearby forested locations around Athens, GA, USA. We identified decaying hardwood logs and extracted adult beetles using hand tools. They were returned to the lab the same day and separated into individual plastic containers containing bits of decaying hardwood, which served as both refugia and a food source. After housing, all beetle containers were undisturbed for 1 week, to allow time to acclimate.
Stressor treatments
Each beetle was exposed to four different mild stressors over a week span, with one stressor applied each day. The different stressor types were applied in a predefined random order, so that the order of the treatments was not the same across all beetles. Testing was done in a lab room with the lights off for three treatments, but lights were on for one (below). On the day of testing, a beetle was removed from its container and placed in a shallow metal sorting tray (40cm x 25cm), which served as a testing arena. An observer applied the treatment of interest and then watched the beetles and recorded their behavior immediately thereafter (below). Following the treatments and observations, beetles were replaced back into their container until the next day of testing, and so on, until each had been exposed to the four treatments. In no particular order, the treatments were light exposure, flip upside down, vibration, and noise + vibration (Fig 2). Exposure to light was conducted when the room lights were on, and this involved merely removing the beetle from its refugia and placing it in the metal tray. We reasoned that this immediate exposure to light (for a beetle that spends its life in logs) would be an adverse stimulus, and elicit freezing behavior. All other treatments were conducted with the room lights off. The upside-down treatment involved placing the beetle on its back in the arena; beetles cannot self-right on a flat surface. The vibration treatment involved placing a cell phone under the metal tray, and using a cell phone app to vibrate the phone and tray. The beetles in the tray thus experienced vibration stimulus, which is known to trigger immobility or startle responses in other beetle species [40][41][42]. The last treatment was the vibration stimulus, plus where the observer sharply rapped 5 times on the side of the metal tray with a metal rod, which we reasoned would serve as an additional vibration and noise stimulus; in preliminary trials we noted that freezing can be induced with just the metal rod (see S1 Video).
Assessing freezing behavior
An observer watched the beetles after each treatment for indications of freezing behavior. From preliminary trials we determined that these beetles react to such threats with an immediate cessation of forward movement, and where the beetle remains in place for an extended period of time (about 20 seconds on average, see results). The head may slowly move from side to side, and there can be slow antennal movement, but no overall body movement. There is no indication that they curl their legs and "huddle", or even stiffen their appendages, which makes us believe this behavior is akin to a "freezing" response, whereby the animals simply stop moving to avoid detection [8]. According to the definition put forth by Sakai [43], this is consistent with a "fear response." It may be that their lifestyle of living in burrows precludes the need to have a "thanatosis" or death-feigning behavior, or that their body morphology is not equipped for this. In fact, we have never witnessed any form of death-feigning in this beetle, after 10 years of study (Davis, pers. obs.). Nevertheless, the freezing behavior was easily observable with training; the observers for both experiments were trained to watch for this behavior and record (with a stopwatch) the start time, and end time, which was when the beetle began moving again (i.e. moving its body away from the initial position in the arena). This duration of freezing was the response variable of interest for our experiments, and in the end we had four recordings for each beetle. Importantly, some beetles showed no freeze response in some trials, and in these cases, we assigned them a zero for the duration. After all beetles had undergone the four different stressor treatments, we computed the average duration for each to use in analyses. Stressor treatments applied to beetles to induce freezing behavior. Beetles were placed in a tabletop arena and exposed to one of four mild stressors, after which we timed the duration of freezing. Stressors included light exposure, flipping the beetle on its back, vibrating the arena (using a cell phone app) and vibration plus rapping the arena with a metal probe (loud sound). Each beetle was subjected to all four treatments on differing days (one treatment per day), and we averaged the durations of all treatments. https://doi.org/10.1371/journal.pone.0281149.g002
Parasite assessment
After the beetles had been tested they were weighed with an electronic balance, and then euthanized with ethanol and dissected to determine levels of parasitism and gender. We carefully removed the abdominal plastron (under stereo magnification) and looked for worms of C. passali, which inhabit the abdominal hemocoel (Fig 1). Beetles can have dozens to hundreds of worms [33,44], and so we used a visual scoring system to simplify nematode assessment. Nematode burden was recorded on a 0-3 scale, where 0 = no worms present, 1 = fewer than 10 worms present, 2 = between 10 and 100, and 3 = more than 100 worms [34,36]. This same categorical scale has been used in multiple prior studies from our lab [35][36][37], and although crude, it does allow for a rapid visual assessment. At the same time, we identified the gender of the beetles based on the presence of the male aedeagus.
Data analyses
Across both experiments we tested 238 beetles. Of these, the final tally of beetles assigned to each nematode group was n = 50, 67, 70 and 51. The duration of freezing variable was log-transformed to approximate a normal distribution. Beetle weight was normally-distributed, based on visual inspection of its histogram. To determine the possible predictors of freezing, we used a general linear model, with predictor variables including experiment number (1 or 2, included as a categorical factor), sex, parasite score (0, 1, 2, or 3, included as a categorical factor), and beetle weight was a continuous covariate. We also included a sex � parasite interaction term. Analyses were conducted using the Statistica 13.1 software package (Tibco Software, Inc.).
General observations
While it was not the focus of the project, we first summarized the overall differences in the beetle reactions to the four different stressors. In the light exposure treatment, 35% entered a frozen state after the stimulus was applied. Meanwhile, 55% of beetles showed a freeze reaction after being flipped onto their back and 56% froze when exposed to substrate vibration. Finally, 59% responded (by freezing) to the combined vibration and noise treatment. The average duration of freezing across each treatment also reflects this variation, though these means include all beetles, even those that did not show any reaction (Table 1). In general, the longest freeze response was elicited by the flipping treatment, though the dual stressor treatment induced the most reactions.
Predictors of freezing
The GLM model that examined all predictors of freeze duration revealed no significant difference between the two experiments (p = 0.7566; Table 2), nor a main effect of beetle sex (p = 0.5449) or parasite load (p = 0.7937). Beetle mass was also not important for explaining variation in freeze behavior (p = 0.1950). Importantly, there was a significant sex � parasite interaction term (p<0.0001), which is illustrated in Fig 3. This graph shows a striking difference in how increasing burdens of nematodes affected male versus female passalus beetles; male beetles tended to increase the duration of freezing as parasite burdens increased, while females appeared to reduce their freezing behavior with increasing levels of parasitism.
Discussion
Horned passalus beetles live with many different types of external and internal parasites [29], though the C. passali nematode is particularly noteworthy for the sheer number of worms that Table 2). Immobility (freezing) is generally associated with boldness/fearfulness, such that bolder or less fearful individuals tend to show short immobility times. https://doi.org/10.1371/journal.pone.0281149.g003
PLOS ONE
Effects of parasites on freezing behavior can inhabit individual beetles. Visually, there appears to be no immune response to the nematodes, since we have never observed melanized or killed worms during dissections (and see S2 Video). However, as nematodes living in the hemolymph, we can reason that the worms must be absorbing nutrients and resources from the blood, thereby limiting what the host can use. In prior experiments in our lab, we determined that this parasite exerts its strongest influence during times when hosts require brief energy bursts, such as during stress events [35,38,39], and wound healing [37], where it (the nematode) appears to moderately reduce host performance. Thus, we undertook this study to ascertain if the nematode similarly influences a behavior that seemingly requires no energy, since by definition, freezing is the cessation of movement (although we did not actually measure energy or metabolic activity during freezing). Further, we did not specifically anticipate that the impact would be positive or negative, since in theory, parasites could cause either scenario, either by causing lethargy (unwillingness to move) or by heightening motivation to forage (to compensate for energy drain) [16]. Regardless of the direction, we reasoned that any effect of this parasite would be consistent across all beetles, and so we were not expecting to discover these distinct differences between sexes in this behavior. In fact, based on our review of the relevant literature on all forms of anti-predator behaviors (thanatosis, tonic immobility, freezing, etc.), we believe this to be the first-described case of a parasite that influences this behavior, and, in different directions within male and female hosts. We are confident that the findings of this project are not due to random chance for two reasons. First, the approach we used-whereby the beetle nematode burden was not known until after all behavior tests were complete-ensured a completely objective and unbiased outcome. Second, we found qualitatively similar results from two different experiments (see S1 File). Interestingly, the fact that similar results were found in our summer experiment, and our winter experiment, also demonstrates that this pattern is not related to the energy cost of rearing and caring for young.
In addition to the reasoning given above, we can also eliminate a wide variety of potential biological explanations, based on prior work in our lab and others where male and female passalus beetles were studied (Table 3). First, male and female beetles appear to be equally Stress behavior Intensity of struggle during simulated attack Females = males [35] Host Physiology Baseline metabolic rate Females = males [37] Host physiology (mass-specific) metabolic rate during wound-healing Females>males [37] Host Physiology Hemocyte density Females = males [38] Host Physiology Heart rate Females = males [45] Host Physiology Heart rate elevation during stress Females = males [45] Host Physiology Water content Females = males [28] � Male-female differences were not reported in the paper but the raw data did indicate no difference.
https://doi.org/10.1371/journal.pone.0281149.t003 parasitized by C. passali, both in terms of prevalence and individual burden [34,44]. Most of the physiological and biological functions evaluated thus far also appear to show that males and females do not differ substantially, especially in terms of energy use; baseline metabolic rate [37] and heart rate [38,45] are both similar between the sexes. The overall feeding rate of males and females (indexed by how much wood is broken down) is also similar between males and females [17], consistent with their similar metabolism. There is no indication that the immune system of males and females differ either, based on hemocyte density [38]. Behaviorally, there are a few sex differences, such as a greater propensity in females to explore than do males [34], and females give more frequent alarm calls than males when attacked. Both of these could be indicators of greater overall boldness in females, though taken with the results from the current study, we now wonder if these prior findings were the result of the parasite, and not the beetle gender per se.
Regarding the behavioral differences between sexes, perhaps the most important indicator to focus on is the freeze reactions of beetles that were not parasitized by C. passali nematodes. It appears that male and female horned passalus beetles may have innately differing styles of dealing with stressors, as noted from the groups with no nematodes (Fig 3). Of these beetles, females appear to remain in a longer freeze state than males (female average = 34 sec, male average = 14 sec), indicating that in that absence of nematodes, females are inherently more fearful, while unparasitized males tend to be more bold. By extension, this could mean that each sex adopts differing means of dealing with predators when parasitized, though the exact mechanism is not clear. Interestingly, the idea that sexes can differ in their reaction to predator stressors is not new; Lagos and Herberstein [47] demonstrated how male crickets have larger elevations in metabolism than females do when presented with predator cues. Similarly, male fruitflies had stronger metabolic responses to a stressor than did females [48]. However, since the opposite pattern was found in our study (female beetles were more fearful), this phenomenon may be species-specific.
Clearly, this project raises many questions that deserve additional investigation. For example, future efforts could investigate whether the worms alter concentrations of any biological compounds within the hemolymph that could affect host behavior, such as amines, which are involved in fight or flight behaviors in insects [49][50][51]. Increases or decreases in concentrations of amines such as octopamine, dopamine or serotonin can affect behaviors similar to what we studied here. Given that the magnitude of changes in freezing behaviors we observed generally matched the intensity of nematode burdens (but differently for males and females), this implies a dose, or concentration effect. That is, more (or less) parasitic nematodes leads to greater host behavioral changes. Therefore, examining amine concentrations of male and female beetles with varying nematode burdens should provide insights.
Despite not yet knowing exactly how the parasite effect arises, we can at least draw inferences or generalities about the ultimate behavior of each beetle sex when heavily parasitized. Given that longer freezing or tonic immobility is generally associated with greater fearfulness [52,53], heavily parasitized male beetles would likely not be motivated to explore or risk exposure to predators by leaving their burrow-either to enter new burrows in their log, or to leave the log entirely in search of a new one. Conversely, our results show heavily-parasitized females appear to be more bold, and intuitively, this means they would be more willing to risk venturing outside their home burrow, or even their log. In fact, this reasoning is consistent with the prior work in our lab, where female beetles were found to be 30% more willing to explore a novel environment than males were [34], and consider also that 70% of horned passalus beetles in nature are parasitized with C. passali.
Given the conclusion above, we believe is possible that this is an example of parasite manipulation of host behavior, designed to improve its transmission [54], which other nematodes are known to do [55]. However, as pointed out by Lafferty and Shaw [16], finding concrete evidence for direct host manipulation by parasites is often elusive. The transmission of C. passali has been suggested (but without evidence) to involve mature larval worms exiting the hosts during the host egg-laying period, which is in the early summer [30]. If this is true, then it is possible that the nematode could be specifically manipulating female beetles to become less risk-averse so they would seek out new burrows or logs to lays eggs in, thereby enhancing its own transmission. However, it is important to point out that C. passali inhabits a region of the body removed from any neural activity (the hemocoel), as opposed to being localized in the brain region where it may be easier to alter neural activity [16]. Though in other nematodes, host manipulation is facilitated by altering serotonin signaling in the host brain [55]. In the end, determining whether this case represents definitive "host manipulation" or an artifact of some inherent host sickness behavior is challenging, and this is problematic in other nematode-host systems too [56]. Moreover, if this were true (C. passali is affecting host behavior), it would also mean that the nematode manipulates male beetles to be more risk-averse and sedentary, for unknown reasons.
To directly test if the pattern we discovered represents actual manipulation of host beetles by C. passali, would require the ability to experimentally infect naïve hosts with the nematode, which we cannot yet do, because of the many unanswered questions around its transmission. Moreover, a confounding issue with using naturally-occurring nematode infections in wild hosts, is that we cannot know for sure the age of the beetles. It is very possible that nematodes build up in individual beetles as they age, and, if age affects host behavior, this could confound interpretation of the "parasite effects" on behavior. This has been shown with species of crickets [57], where males became less bold as they aged, but females did not. The authors of that paper thought this was due to the predation risk associated with calling for mates (which is not the same in this system). From a logistical standpoint, conducting such an investigation on age effects would be difficult in our beetle system. This would require having known-aged beetles to test, which could only be possible by rearing them in captivity, and this alone is difficult with this species, since young grubs are raised by their parents [33]. Second, and again, we do not yet know how to experimentally infect hosts with this nematode.
More generally, these results highlight an important knowledge gap in the body of research around anti-predator behaviors, which is the role of parasites, and how they have the potential to influence the host reactions to predators or related threats. To our knowledge, no prior studies of this topic have considered this potential factor, either in the study design, or when interpreting results. Indeed, in a thorough review of the topic (of 91 studies), no mention was made of this [8]. Further, the knowledge that parasites can even alter behaviors of males and females differently, could also have implications for other projects where anti-predator behaviors have been compared between sexes [58,59]. Given that ours is the first study to bring this issue to light, this is clearly a nascent topic needing of more exploration.
Conclusions
We exposed wild-caught horned passalus beetles to four different mild stressors and recorded how long they freeze (cease movement) in response. After pairing these data with the beetle nematode burdens, we discovered that heavily parasitized female beetles reacted much differently than heavily parasitized males to the same stressors. Female beetles with heavy nematode burdens appear to be more bold than similarly-parasitized males, which itself engenders questions about how this affects their annual cycle. There are few known physiological differences in the beetle sexes that could explain the sex difference, and therefore more investigation is needed to fully elucidate the mechanism. This discovery highlights the importance of understanding the impact of parasites to anti-predator behaviors in animals, a topic which has been neglected.
Supporting information S1 Video. Video segment showing a horned passalus beetle freezing briefly after an observer gently rapped on the tray. (MP4) S2 Video. (MP4) S1 File. Document containing figures that display results from each experiment separately. (DOCX) S1 Raw data. Excel file containing complete dataset generated from this study. (XLSX) | 2023-03-15T06:18:13.649Z | 2023-03-14T00:00:00.000 | {
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220792229 | pes2o/s2orc | v3-fos-license | Activity: A Basic Teaching Form of Morality and Rule of Law Lessons in Primary Schools
As a basic teaching form of morality and rule of law lessons in primary school, activity plays a significantly importance role in mobilizing the students’ enthusiasm and subjective initiative. By participating in a variety of activities, students can improve their knowledge structure and enrich their life experience, thereby promoting the formation of students' correct concepts and good qualities. There are massive activities which can be designed by teachers to trigger students' creativity and enhance their practical ability. In this paper, methods, openness, integration of activities as well as how to conduct the activities are investigated. In general, the activities of morality and rule of law lessons in primary schools mainly include: observation activities, investigation activities, discussion activities, visit activities, production activities, planting activities, breeding (feeding) activities, exchange activities, participation in ceremonial activities, filial piety activities, environmental protection activities. These activities can be arranged before, during or after class by combination with other activities, such as class activities, school activities, festival activities and family activities. Based on different themes of teaching, it is suggested that teachers can conduct these activities by direct activities or communication activities. It is widely acknowledged that the activities of a class often exist in a comprehensive way. The development of activities can achieve the best teaching results by taking the teaching content and the actual situation of the class into consideration.
Introduction
In recent years, as an important part of primary school education in China, morality and rule of law lessons are prominent in many courses. Teachers should focus on the actual life and psychological needs of primary school students, and solve the relationship with themselves, with others, with the collective, with the country and society [1][2]. With a view to the healthy growth and all-round development of students, teachers should take on the educational mission given by the times [3][4].
A notable feature of morality and rule of law lessons is focusing on the connection between students' life experience and social practice. The aim of this course is mainly to provide students' experience, perception and automatic construction during the activities under the guidance of the teachers. By participating in a variety of activities, students can evidently improve their knowledge structure and enrich their life experience, thereby promoting the formation of students' correct concepts and good qualities [5][6].
Therefore, in classroom teaching, teachers are supposed to follow practical principles, take students as the foundation, and design classroom practical activity programs around the problems in students' lives, so as to help students profoundly understand and master the requirements and norms of social life [7]. Excellent practical activity design can not only help stimulate students' interest in learning and attract students to participate in active inquiry, but also promote the formation of their good behavior habits [8]. As a result, the four basic characteristics of morality and rule of law lessons consist of activity, daily life, comprehensiveness, and openness. The activity has also become the basic form of teaching and learning in moral and rule of law courses. In this paper, methods, openness, integration of activities as well as how to conduct the activities are systematically investigated.
Methods of Activities
Considering that there are different contents and meanings, activities can be classified into several kinds. Generally, the activities of morality and rule of law lessons in primary schools mainly include: observation activities, investigation activities, discussion activities, visit activities, production activities, planting activities, breeding (feeding) activities, exchange activities, participation in ceremonial activities, filial piety activities, environmental protection activities. Through these methods, students interact with the environment and collaborate with their classmates to gain personal experience and feelings about themselves, others, nature and society, and hence triggering their creativity and enhancing practical ability effectively.
It should be noted that the left behind children (students) in rural areas caused by population flow has become a social problem that cannot be ignored [9][10]. Although the number of the left behind children in rural areas in China has gradually decreased in recent years, the number of left behind children in rural areas still exceeded 10 million in 2017, as displayed in Figure 1. The decrease of parents' company and neglect of education are the biggest reasons for most left behind children to become problem children. Many parents are going closer to their children with the economy slows and more and more cities tries to make more jobs in the inland economy [11][12]. That means they can see their children as often as every few weeks, rather than only once a year or less. And technology can put parents in touch with their offspring relatively inexpensively. From the perspective of these left behind children, however, active participation in different activities can efficiently reduce their loneliness and help them interact with peers while building correct outlook on life, value as well as the world.
Openness of Activity Time
According to the arrangement system of morality and rule of law lessons in primary schools and children's cognitive laws, activities can be arranged before the class. For example, based on the textbook "Morality and Rule of Law" approved by Chinese Ministry of Education in 2017, students from Grade Two are encouraged to plant a peanut with the guidance of their parents, and they can also plant sprouted potatoes and flowers purchased online. Through planting activities, they can learn to plant correctly under the guidance and communication of family members and classmates, learn to write planting notes, experience the process of plant growth, and then love the fruits of others' labor and cherish the life of plants. When teaching this lesson, the activity becomes communication activity between teachers and students. If there is no planting activity before the course, the curriculum objectives are not achieved in children's experience, perception, and active construction, making the teaching activities in this lesson impossible.
Activities can also be carried out during the class. For instance, teachers and students can participate in the activities of greeting cards and caring walls arranged in "New Year's Gifts" class (the 16th class of the textbook "Morality and Rule of law" approved by Chinese Ministry of Education in 2016). In this kind of production activities, students can learn to write blessings in accordance with their identity and raise hopes according to different objects.
Likewise, activities can be conducted after class. In the "Doing housework" class, the activities can be changed in time and place. There is another trick that teachers can arrange for the children to return to their own home to have an "exam". Students are inquired to take out their own clothes and review the method of folding clothes, which is instrumental in obtaining practical ability and rich life experience.
Flexible scheduling of activity time is conducive to opening the barrier between family, community and classroom. The non-enclosed class model helps students broaden their horizons and enrich their mind.
Integration of Activities
Based on different life situations/activities, moral and rule of law courses can be divided into different areas, as illustrated in Table 1. Studying in different fields can help students form a good outlook on life. It is a continuous development process for student to form their character and behavior habits and accumulate knowledge and experience in their lives [13]. As a result, strengthening the integration and continuity between educational activities is essential for improving the effectiveness of the curriculum. The activities of morality and rule of law lessons should be combined with other activities that students involved in, as shown in Figure 2. (1) Class activities. There is no doubt that it should be combined with class activities, such as class rules and dressing up the classroom. By combining with the class adviser, the teachers can guide the students to participate in these activities to maximize the effect of the teaching activities.
(2) School activities. It should be combined with school activities, such as caring for public property, not throwing things around, learning to line up and so on. In school activities, the effect of teaching activities on the habits of students can be checked effectively. Through the school activities, feedback on the effect of teaching activities can be obtained to further guide and teach the students. The courses can continuously influence children's real life by combining the requirements of teaching activities as well as school activities.
(3) Community activities. In general, there are some similarities between safety education activities, theme recreational activities and morality and rule of law. Teachers can take advantage of these similarities and combine them with community activities so that children can gain deepening experiences.
(4) Festival celebration. Teachers can use national day to educate and guide children to strengthen patriotic feelings and establish lofty aspirations. At present, many parents and teachers tend to pay more attention to intellectual education than moral education for students' growth and regard patriotic education for them as a matter of school, which is wrong. Class is the cell of society and the social window of life. The quality of patriotic education directly affects the healthy growth of children. Therefore, students have responsibilities and obligations to do a good job in patriotic education for their students. Also, students can understand the origin and customs of the Mid-Autumn festival and master and understand the poems and verses about the Mid-Autumn Festival.
(5) Other disciplines. The activities of making greeting cards, as mentioned in section 2, can be integrated with the art discipline. The greeting cards made by students can be more artistic, appreciative and collectible. Our life in Qingming can be also integrated with information technology course. Through the online activities of sacrificing heroes, the memorial ceremony for ancestors is organically combined with the love and reverence for heroes, which is beneficial for cultivating students' patriotic feelings and spread positive energy.
(6) Family activities. Students spend a long time in the family, have blood and family ties with their families while having family members to accompany and guide them in activities. Family education and school education can also be well combined. Children can go to school with the company of their families, so as to understand what is on the road, what needs attention, how children think and what their families require. Through the "stampede" activities with family members, communication activities in the classroom, and discussion activities among students, children will understand that there are various dangers on the way to school and establish a sense of safety.
Direct Activities
According to the theme of teaching, teachers can directly arrange students' activities, so that students can experience in the activities. In activities, children's emotions, attitudes, cognitive abilities, moral habits and behavior habits, learning methods and characteristics are fully displayed and gradually formed. These activities include the making of greeting cards, the experience of the meaning of ringing tones, the arrangement of classrooms, the singing of the national anthem, the poetry recitation activities related to the Mid-Autumn Festival, etc. Such activities account for the vast majority in morality and rule of law lessons.
Communication Activities
Communication activities are arranged before class and the activity cycle is relatively long. Students gain a lot through speculation, imagination, observation, record and discovery. After activities, students can share their findings and experiences with others in class under the supervision of the teachers. This kind of activity comprises the planting and breeding activities as well as the way students go to school accompanied by their families. If the condition permits, students may take photos and make slides to make the class more attractive.
Activities are of importance in morality and rule of law lessons in primary schools, but they can't be just a simple form. Activity design by teachers must be based on the students' real life and attack their enthusiasm, making the classroom alive.
It is widely acknowledged that the activities of a class often exist in a comprehensive way [14][15]. The development of activities can achieve the best teaching results by taking the teaching content and the actual situation of the class into consideration.
Conclusion
In this study, methods, openness, integration of activities, as well as how to conduct the activities are systematically investigated. Based on the results, following conclusions are achieved: (1) The activities of morality and rule of law lessons in primary schools mainly include observation activities, discussion activities, visit activities, production activities and so on.
(2) These interesting activities can be arranged before, during or after class by combination with other activities, such as class activities, school activities, festival activities and family activities.
(3) Based on different themes of teaching, it is suggested that teachers can conduct these activities by direct activities or communication activities. The development of activities can achieve the best teaching results by taking into account the teaching content and the actual situation of the class. | 2020-07-26T19:57:42.145Z | 2020-07-22T00:00:00.000 | {
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16995280 | pes2o/s2orc | v3-fos-license | FH Leonis, the first dwarf nova member of a multiple star system?
Sudden brightenings of HD 96273 or BD+07 2411B were observed with the HIPPARCOS satellite in 1992,which still require a convincing explanation. A new analysis of all known data of these two stars is given, including additional information on the Balmer line equivalent widths. The brightenings can be explained as SU UMa type dwarf nova outbursts, superimposed on the combined light of two normal F and G type main sequence stars. Since the hypothetical dwarf nova turns out to be located at the same distance as HD 96273 and BD+07 2411B, we possibly see here the first case of a cataclysmic variable as a member of a multiple star system. Questions on history and evolution, as well as possible ways to confirm this interpretation, are briefly outlined.
Introduction
A recent paper in this journal, by Dall et al. (2005, hereafter DSSI) was titled "Outbursts on normal stars -FH Leo misclassified as a novalike variable". The authors report three possible brightenings of the combined flux of the wide binary HD 96273 and BD+07 2411B observed with the HIPPARCOS satellite in 1992, January 3, January 15 and June 19 with amplitudes between 0 m .1 and 0 m .35 above the normal brightness level of this double star. They obtained high resolution spectra of both stellar components, determined their effective temperatures, surface gravities, microturbulence parameters, rotation and radial velocities as well as abundances of several chemical elements. Since both stars behave -according to all these parameters-like normal main sequence dwarfs, DSSI concluded that the identification as a cataclysmic variable must be wrong. They discussed several possible causes of the observed brightenings, in particular transient background or foreground objects, magnetic interaction with an unseen companion, a planetary accretion event and a microlensing event. Finally, they point out that none of these explanations seems to be convincing.
In this Research Note I would like to show that most of the observed properties of FH Leo can have their natural explanation if we assume that it is a dwarf nova at the same distance as HD 96273 and BD+07 2411B.
FH Leo as a dwarf nova companion of HD 96273 or BD+07 2411B
The HIPPARCOS distance of this binary is 117 pc. This implies an absolute magnitude M V = +3 m .6 of HD 96273 and M V = +4 m .8 of BD+07 2411B. The effective temperatures determined by DSSI correspond to spectral types F6V (HD96273) and G3V (BD+07 2411B). These abso-Send offprint requests to: N. Vogt, e-mail: nvogt@ucn.cl lute magnitude values and also the observed colours are in accordance with a classification of a normal main sequence star, the interstellar reddening can be neglected. The combined absolute magnitude in the blue spectral range is M B = 3 m .76. Since on 1992, January 3 (JD 2448624) a brightening of 0 m .27 on average was observed, the absolute magnitude of the total light during this event was M B = +3 m .49. Therefore, the absolute magnitude of the brightening source alone is M bs = +5 m .13, which is nearly identical of that of a dwarf nova at outburst maximum: equation (3.4) of Warner (1995) predicts a value between +5 m .2 and +5 m .3 for a dwarf nova with an orbital period between 1.5 and 2 hours. On 1992, January 15 (JD 244 8636) the absolute magnitude of the brightening source was M bs = +6 m .1, about 1 m fainter than 12 days before. A natural explanation of this behaviour is that of an SU UMa type dwarf nova which begun its superoutburst in 1992, January 3 and declined slowly towards the end of its plateau phase in January 15. This way, even the differences of about 0 m .1 between the brighter and the fainter magnitude measures in January 3 could be explained by the superhump activity; the brighter points may refer to the superhump peaks, their time difference is about 2 hours, compatible with the superhump period of a typical SU UMa type dwarf nova. The total intrinsic superhump amplitude would be of the order of 0 m .4, just as expected during the early stage of a superoutburst.
One important question, however, remains, the absorption spectra of HD 96273 and BD+07 2411B look rather normal. In particular, DSSI did not find any traces of hydrogen emission which would be typical for dwarf novae in quiescence. For instance, Thorstensen et al. (2002) give Hγ emission equivalent widths of 40Å for V844 Her and 20Å for DI UMa. In Hβ even values up to 100Å are possible (Warner, 1995, Fig. 2.35). Indeed, the hydrogen emission may be present in FH Leo, but unobservable due to the large difference in brightness between HD 96273/BD+07 Nikolaus Vogt,: FH Leonis, the first dwarf nova member of a multiple star system? 3 2411B and the hypothetical dwarf nova in quiescence. If FH Leo is an SU UMa type dwarf nova with a long outburst cycle, similar to WX Cet (Sterken et al., 2006) we expect an amplitude ≥ 6 m , revealing an absolute magnitude ≥ 11 m .1 of the dwarf nova in quiescence. Because of the large difference in the continuum flux, even in the blue spectral range, an emission equivalent width of Hγ of 40Å of the quiescent dwarf nova would be reduced to a contribution of about 0.045Å in the equivalent widths of HD 96273 or BD+07 2411B while the above maximal EW value of Hβ would contribute 0.11ÅḊSSI did not publish their measured equivalent widths of these stars, but the authors kindly sent me an original spectrum of each star. From this I have determined the equivalent widths of Hα, Hβ, Hγ and Hδ. Their values are 3.7±0.2, 5.9±0.3, 7.4±0.8 and 5.1 ± 0.3 resp. for HD 96273 and 3.0 ± 0.2, 3.6 ± 0.3, 5.5 ± 1.0 and 3.2 ± 0.2 resp. for BD+07 2411B (the given errors refer mainly to uncertainties in the continuum determination. An emission contribution of the dwarf nova in quiescence would reduce such equivalent widths by a very small amount (≤2%) which certainly is impossible to recognize even in high resolution spectroscopy. Therefore, it is not surprising that DSSI did not find any anomaly in the spectra of HD 96273 and BD+07 2411B.
The brightening event of 1992, June 19 (JD 2448793), about 169 days after the "superoutburst" detection, could perhaps be explained as a short outburst of the dwarf nova. However, there are some other measurements without a significant brightening, only a few hours before and afterwards. Therefore, this event could also refer to a short-lived "flare" which also was observed in other dwarf novae (Bateson 1989). In any case, further observations will be necessary to confirm the existence of short eruptions or flares in FH Leo.
Conclusions
The most plausible explanation of the outburst behavior of FH Leo would be that of an SU UMa type dwarf nova, superimposed on the wide binary HD 96273 and BD+07 2411B, and placed at the same distance as this binary. If this is true we either have a very rare and improbable chance coincidence, or simply a gravitationally bound multiple star system with a dwarf nova orbiting HD 96273 or BD+07 2411B. To my knowledge, this would be the first case of the detection of a cataclysmic variable as component of a multiple star system. Rather interesting questions would arise concerning the possible evolutionary history of such a system: What is the age of it? What was its origin and how did it evolve? Since the HD 96273/BD+07 2411B system is a rather wide binary (projected physical separation 936 AU) there is plenty of space for hierarchical sub-structures of smaller dimensions, such as a giant-type progenitor of a cataclysmic variable which has to pass the subsequent common envelope phase.
This interpretation, however, needs to be confirmed. This can be done in different ways: First of all, we will monitor FH Leo in the next season closely and search for additional outbursts, which also should reveal superhumps, the main characteristic of any SU UMa type dwarf nova. This will be done in our Cerro Armazones Observatory (Universidad Catolica del Norte, Antofagasta) and possibly at other institutions. On the other hand, we will search for outbursts of FH Leo in the patrol plate archive of the Sonneberg Observatory, which is, following Harvard, the world-wide second largest in size and plate number (Braeuer and Fuhrmann 1992). It covers several decades beginning about 1930, and was used to detect more than 10000 new variable stars. The typical threshold of detection with blink comparator or similar visual inspection methods, as applied traditionally at the Sonneberg Observatory, is about 0 m .3, just the total amplitude of FH Leo. Therefore, it is not surprising that FH Leo remained undetected until recently. New scanning and reduction methods as applied by Vogt et al. (2004), however, allow us to reduce this threshold to less than 0 m .1, thus enabling the detection of historical outbursts of FH Leo. A search for them is in preparation. | 2014-10-01T00:00:00.000Z | 2006-03-27T00:00:00.000 | {
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17004722 | pes2o/s2orc | v3-fos-license | Naproxen Twice Daily Versus as Needed (PRN) Dosing: Efficacy and Tolerability for Treatment of Acute Ankle Sprain, a Randomized Clinical Trial.
PURPOSE
This study was conducted to compare the efficacy and safety of naproxen 500 mg twice daily (BID) versus naproxen 500 mg as needed (PRN) for treatment of ankle sprain.
METHODS
In this seven-day, randomized, parallel group trial, 135 patients with ankle sprain occurring less than 48 hours prior to the first dose of study medication were randomized to receive naproxen 500 mg BID (67 patients) and naproxen 500 mg as needed (PRN) (68 patients). The ankle pain was assessed at rest and on full weight bearing using Numeric Rating Scale (NRS) from 0 (no pain) to 10 (the worst imaginable pain). Ankle swelling was assessed as a 4-point scale ranging from 0 (no swelling) to 3 (severe swelling) rated by the investigator. The primary efficacy end point was the patient's assessment of ankle pain via NRS and the degree of swelling on day seven.
RESULTS
Results showed a significant decrease in pain on weight bearing, pain at rest and the extent of swelling (P<0.001) in both groups, but there was no substantial difference between the two groups (P>0.05) after seven days. Assessing the safety profile of the two different dosing, 13.3% of the naproxen BID group and 6.7% of the as needed group had adverse events, showing that the as needed regimen was safer (P<0.001).
CONCLUSION
Results showed that naproxen as needed may reduce the pain and edema of the sprained ankle with no significant difference compared to the BID regimen, while it possesses better safety profile and lower total drug use.
INTRODUCTION
nkle sprains are among the most common musculoskeletal injuries that occur in everyday life and sports related activities [1][2][3] . Approximately one ankle sprain occurs per 10000 person everyday [2,4,5,6] . Inadequate and incorrect management of ankle sprain can result in prolonged complications such as decreased range of motion, chronic pain, early degenerative bony changes and chronic joint instability [7] . Ankle sprains are classified as mild (first-degree), moderate (second-degree), or severe (third-degree) according to the extent of pain, swelling, tenderness, joint instability, ecchymosis, functional loss and difficulty in walking [8][9][10] . Initial management goals are to limit inflammation and swelling and to restore normal function as much as possible [11] . Conventional treatment for ankle sprains includes the 'rest, ice, elastic compression and limb elevation' (RICE) protocol, protected weight bearing, early ambulation and use of analgesic and antiinflammatory drugs [6,8] . Non-steroidal anti-inflam-A matory drugs (NSAIDs) such as ibuprofen, naproxen, diclofenac and piroxicam effectively reduce the pain and swelling associated with sprains [6][7][8][9][10][11][12] . No particular NSAID has superiority over the others for the treatment of ankle sprain [8] . NSAIDs also inhibit platelet aggregation, which has an important role in healing of the wound and gradual fading of the ecchymosis [10] . However, these medications have several adverse effects such as gastrointestinal upset, gastric and duodenal ulceration, perforation and hemorrhage [11] . NSAIDs' adverse effects increase with upper dosage and duration of use. To the best of our knowledge, none of the published studies have compared the regular versus intermittent (as needed) dosing of such medications [7] .
The aim of this study was to evaluate the efficacy and safety of the "as needed" dosing of naproxen compared to the twice daily dosing in the management of grade 1 and 2 acute ankle sprain.
METHODS AND SUBJECTS
This was an open label, randomized, outpatient, activecontrolled, parallel-group, clinical trial conducted at emergency department (ED) of Imam hospital, Tehran, a general teaching hospital with an annual census of 40,000 visits. The study was conducted between May 2009 and October 2010. Inclusion criteria were as follows: the patients were aged ≥ 18 years and presented with an isolated unilateral mild to moderate soft tissue injury of the ankle (first-or second-degree ankle sprain), which had happened 48 hours prior to the first dose of medication. Also, the patients were included if they mentioned pain score of 4 or above on a verbally administered, 11-point numeric rating scale (NRS). All women of child bearing age had to have a negative urine pregnancy test, and not to be breast feeding.
Patients were excluded if any of the following criteria were present: preexisting ankle problems (including osteoarthritis, fracture, sprain, congenital deformity); a similar injury of the same joint within the past six months, presence of bilateral ankle sprain, third-degree sprain or ipsilateral knee injury; radiographic evidence of fracture or syndesmosis injury; known history of significant renal impairment (creatinine level >160 µmol/L) or hepatic insufficiency (aspartate aminotransferase >54 U/L or alanine aminotransferase >42 U/L); lower limb thrombosis; diabetes mellitus; chronic or toxic alcohol ingestion; known severe congenital or acquired coagulopathy; active gastrointestinal disease; history of esophageal, gastric or duodenal ulcer; sensitivity or allergy to NSAIDs; treatment with an intra-articular injection of a corticosteroid or hyaluronic acid in any joint within eight weeks of the first dose of study medication; treatment with any oral or intramuscular corticosteroid within 30 days of the first dose of study medication; use of non-COX-2 selective NSAIDs, COX-2 selective inhibitors (except aspirin ≤ 325 mg/day for cardiovascular prophylaxis), or other medications such as neuroleptics, tricyclic antidepressants, and lithium that could potentially confound the assessment of analgesia within 24 hours of the first dose of study drugs; pregnancy; history of current or past psychiatric disorders; or inability to return for follow-up.
All patients were informed of the nature and potential risks of the study and they provided written informed consent to participate. The research protocol was conducted in accordance with the ethical principles of the Declaration of Helsinki.
Immediately after arrival at the ED, the patients were visited by research associates (emergency medicine residents or attending physicians). Before enrollment, patients underwent a baseline assessment including history and physical examination. The extent of ankle swelling, pain, and stability were evaluated by the same physician: The ankle pain was assessed at rest and on full weight bearing using NRS from 0 (no pain) to 10 (the worst imaginable pain). Ankle swelling was assessed as a 4-point scale ranging from 0 (no swelling), to 3 (severe swelling). In order to assess joint stability, clinical tests including ankle squeeze test (indicator of injury to anterior talo-fibular ligament), external rotation stress test (representing injury to syndesmosis), anterior drawer test and talar tilt test were performed. Based on the above parameters on physical examination, investigators classified the ankle sprain as first-degree, second-degree or third-degree.
First-degree ankle sprain is defined as partial tearing of the lateral ligaments complex, while the stability and the structure of the joint are completely preserved. On physical examination, the pain on weight bearing is little and not severe, thus the patient has full weight bearing; while the edema is rare and ecchymosis does not occur. In second-degree ankle sprain, tearing of the anterior talo-fibular ligament occurs, while the calcaneo-fibular ligament is preserved. In addition to point tenderness and partial instability of the joint, the pain is moderate to severe on weight bearing. Ecchymoses occur frequently and the edema is also moderate to severe. In third-degree ankle sprain, the patient is disabled while the joint capsule is disintegrated and the joint is unstable. The reliability of this clinical grading system for the sprained ankle has been previously approved [2] . After initial assessment, inclusion and exclusion criteria were applied and eligible patients were enrolled. Baseline data were collected (including patients' name, gender, age, occupation, mechanism of injury, and time since the injury occurred) and recorded in the first visit form.
After completing assessment, the research associates randomly assigned patients to one of the two treatment groups to receive either naproxen 500 mg twice daily (BID) or naproxen 500 mg as needed (pro re nata, PRN) for pain but not more than twice daily. Block randomization was applied using a computer generated random sequence of numbers in 4-digit blocks. Treatment duration was seven days, after which the patients were re-assessed for pain severity and ankle swelling as well as for occurrence of any adverse events by the same physician. Both groups received 14 naproxen tablets (Iran Najo Co., pharmaceutical hygienic and cosmetic, Tehran, IR Iran) at the first visit and were asked to return the remaining pills for the follow up visit. They were instructed when and how to use the drug according to the study protocol and were asked not to use other analgesic medications during the study period. The 'rest, ice, and limb elevation' protocol was recommended for all patients. For immobilization, a short-leg splint was applied for all patients. Patients were requested to keep their splints until the follow-up visit and elevate the injured leg for 48 hours. Non-weight bearing was recommended until they could walk with a normal gait and no pain.
In the follow-up visit, set on day seven, assessments by the same clinician included ankle pain scores at rest and on full weight bearing, and ankle swelling. Participants were enquired about the adverse effects of the study medications. With regard to adverse effects of naproxen, prevalent major and minor adverse effects such as gastrointerstinal bleeding or upset were investigated. Data were recorded in the follow-up visit form. The patient's compliance was calculated using this formula: Where "n" was the number of pills returned. The primary measures of efficacy included changes in pain during weight bearing and at rest from baseline visit to the follow-up visit and changes in ankle swelling. Secondary measures were the proportion of patients whose pain score decreased by at least two on the NRS at the follow-up visit, as well as the rate of adverse events.
The sample size of about 67 patients for each group was determined according to the day seven rest pain NRS score (difference of less than two) to show comparable clinical effect of two different dosing schedules.
Data were analyzed by SPSS Windows 10.0.5 (SPSS, Chicago, IL). Significance level was considered to be < 0.05. Parametric Student's t-test was used to compare the characteristics of the patients in the two groups. The Mann-Whitney test compared differences in ankle swelling and the paired t-test was used to compare differences in rating of pain at rest and on weight-bearing within each group and between day 0 and 7. Differences in the rate of drug adverse events between the two groups were compared using Fisher's exact test.
Since the medication (i.e. naproxen) was the same in both groups and the study intervention was different dosing of the medications, the patients could not be blinded to the study intervention. Before the allocation of a patient to a study protocol his or her data were collected and recorded, so blinding of the investigator was not needed for the first visit. For the follow-up visit, the parameters were assessed with the same, most of which were objective measures.
Subjects:
Of the 155 patients screened, 135 were considered eligible for enrollment and were randomized to receive either naproxen 500 mg BID (67 patients) or naproxen 500 mg as needed (PRN) for pain (68 patient). Five patients from BID group and eight patients from PRN group did not take the study medication or did not refer for follow up and eventually 122 patients completed the study protocol and were included in our analysis (Fig. 1).
Baseline characteristics:
There was no significant difference in baseline characteristics between the two study groups (Table 1). According to table 2, there was no significant difference between the two groups in baseline pain score measured through NRS at rest, baseline pain in daily activities (on weight bearing) and ankle swelling.
Primary measures of efficacy:
Results from the follow-up visit showed a significant decrease in pain on weight-bearing on day seven with mean decrease in pain scores of 4.8 from the baseline (P<0.001). The overall pain decrease on weight bearing as well as the decrease in pain at rest was not significantly different between groups. Swelling decreased significantly during the seven days trial (P<0.03 in BID group and 0.003 in as needed group) but there was no substantial difference between the two groups ( Table 2). Planed analysis showed that naproxen 500 mg as needed was not inferior to naproxen BID in reducing the baseline primary measures of efficacy.
Assessing the safety profile of the two different naproxen dosings (table 3), the as needed regimen was safer (P<0.001) compared to the BID regimen. There were no serious adverse events (i.e. gastrointestinal bleeding) or death ( Table 3).
The mean numbers of tablets returned by BID and as needed groups were 3.5 and 9.4 tablets, respectively. In this case, we observed a significant difference between adherence to therapy in both groups (P<0.01).
DISCUSSION
This study used ankle sprain as a model of acute musculoskeletal pain to evaluate the safety and efficacy of Naproxen 500 mg BID or Naproxen 500 mg as needed for the management of acute musculoskeletal pain. We used the 'Rest, Ice, elastic Compression and limb Elevation' (RICE) protocol for all of the patients in both treatment groups. Grade 1 and 2 ankle sprains show a good clinical response to non-operative management (RICE protocol) [13,14] . However, the best conservative treatment, both in costs and clinical outcome is not clarified in present clinical trials.
Non-steroidal anti-inflammatory drugs (NSAIDs) are more effective than placebo for the initial treatment of ankle sprain [15,16] . Naproxen 500 mg BID is effective in the treatment of ankle sprain and other soft tissue injuries, and has a good safety profile [9,17] . Cukiernik and her colleagues compared acetaminophen versus naproxen in the treatment of ankle sprain in children [9] . Both drugs were equally effective in reducing pain and disability. They suggested that as needed dosing of NSAIDs in the management of soft tissue musculoskeletal injuries should be further studied [9] . In our trial, also, naproxen BID proved to be effective in reducing ankle swelling, pain on weight bearing and pain at rest with no serious adverse events. However, 8% of patients treated with naproxen BID reported some minor complications mostly gastrointestinal upset. This was lower than the report by Kyle et al (23%). They compared naproxen 500 mg BID with lumiracoxib in the treatment of acute musculoskeletal pain, and concluded that both drugs were similarly effective in reducing pain intensity during the five-and seven-day periods [18] .
Patients treated with naproxen as needed, showed the same clinical result as naproxen BID group, but adverse events reported by this group and the number of pills used were significantly lower. To our knowledge, higher doses of NSAIDs increase the risk of GI complication. Warner and Mitchell performed a systematic review and demonstrated a correlation between NSAIDs use and hospital admission due to perforation or hemorrhage [19] . They found that lower GI complications risk of ibuprofen is due to the lower dosage of the drug used in general practice. They concluded that "Use of low risk drugs in low dosage as first line treatment would substantially reduce the morbidity and mortality due to serious gastrointestinal toxicity from these drugs" [19] . Rodríguez and his colleagues conducted an analysis of users and nonusers of acetaminophen and NSAIDs. They concluded that acetaminophen doses higher than two grams and sustained-release formulation of NSAIDs were associated with higher incidence of GI complications [20] .
Our study has several limitations; the study participants were mostly among the low to middle socioeconomic population group and accordingly, we had lost several patients to follow-up. A significant proportion of patients did not fill the diary about their pain and time they used the analgesic medication, so we could not conclude that in which group the pain free situation had occurred sooner. Furthermore, the fact that our patients had different adherence to therapy -as a secondary measure of our study-might have indirectly influenced the decrease in pain scores. With regard to adherence, we could not collect data regarding the amount of pills consumed on each day after injury in the PRN group, which might have considerably influenced the extent of pain decrease among the patients. Additionally, we did not consider the use of RICE protocol by patients prior to their referral to our center. Also, it would be better to perform at least two or three follow-ups in order to evaluate the back-to-work time interval of patients. We suggest future studies consider a third control group which would receive no treatments other than RICE.
CONCLUSION
Our study showed that naproxen as needed may reduce the pain and edema of the sprained ankle with no significant difference compared to the BID regimen, while it possesses better safety profile and lower total drug use; therefore, we recommend physicians to prescribe naproxen as needed for pain instead of the regular twice daily doses. | 2018-04-03T01:07:17.815Z | 2013-08-02T00:00:00.000 | {
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235666513 | pes2o/s2orc | v3-fos-license | Understanding gas prices: an overview of regulations and components affecting the Indonesian natural gas prices
Natural gas is the primary commodity of Indonesia’s energy supply and an important policy issue. Domestic gas demand is dominated by the industrial, power sector, and fertilizer. Over the past five years, Indonesia’s natural gas prices have increased significantly, which will undoubtedly affect the consumers. We summarized and analyzed policy, literature, and interviews with stakeholders to find the main factors of gas prices. This study concluded that the government has a more dominant role in determining gas prices. The increase in gas prices was mainly influenced by the upstream project’s economics, the tariff for transmission, and managing natural gas infrastructure. The application of indexation with ICP and the escalation price also contributed to the high price. The Government needs to consider gas price adjustments carefully because these commodities have a multiplier effect and affect industry competitiveness.
Introduction
Natural gas is the third primary energy used in Indonesia after oil and coal [1]. Natural gas has the advantage of being abundant, environmentally friendly, and an efficient source of energy, if it is compared to other energies [2]. In electric power generation, natural gas offers lower construction and fuel costs. For the fertilizer and petrochemical industry, natural gas is a raw and supporting material that has many advantages. Likewise, households that receive gas through the city gas program have felt the positive impact. The economic policy shifted the natural gas paradigm, which was previously a commodity that becomes national economic growth, added value creation, and labor absorption [3].
The price of natural gas affects the upstream oil and gas industries and industrial use. The determination of the gas price of 6 US$/MMBTU through Presidential Decree No. 40 of 2016 is troublesome for upstream oil and gas contractors (Contractors of Cooperation Contract/KKKS). On the other hand, the price of natural gas as fuel contributes 23% of the total production cost in the steel industry [4], even when it functions as raw material, the portion can reach 70% as in the fertilizer industry [5]. Indeed, the price of natural gas determines the price and competitiveness of industrial products.
A good understanding of natural gas pricing is essential for business actors because it is helpful for business decisions. This paper provides a systematic, comprehensive content analysis and literature study. The researcher serves to study from all binding regulations, literature studies, and in-depth investigation.
Natural gas market
In this section, the author will review the natural gas business chain in Indonesia. Each stage of the business chain impacts the natural gas price.
Indonesian gas utilization
Currently, Indonesia still exports natural gas in the form of LNG and piped gas. Natural gas is a basic need in increasing industrial and economic activities. Then, economic growth, Indonesia's natural gas market began to grow and shifted exports. One of the National Development agendas contained in the 2020-2024 RPMJN, the Government will strengthen economic resilience by increasing the use of domestic natural gas. Domestic use of natural gas has increased by an average of 7.8% annually since 2005, and in 2018 it reached around 60% of total production [7]. The Government targets domestic natural gas utilization increasing from 2014 by 19% of the total national energy mix to 24% in 2050 [3].
Indonesia gas balance and natural gas infrastructure
Indonesia's natural gas reserves as of January 2019 reached 77.29 TSCF [7]. In keeping the domestic gas supply, the Government and upstream oil and gas contractors are trying to develop several upstream natural gas projects, including East Natuna, Jambaran Tiung Biru, IDD, Tangguh Train 3, Asap-Kido-Merah, and Abadi Field (Masela) [8]. However, some of these upstream natural gas projects are far from demand or customer need and even offshore so that later it will affect prices at the end consumers.
The utilization of natural gas is very dependent on infrastructure as well as the transmission and distribution network that connects gas sources to consumers. Infrastructures are the key to meeting Indonesia's gas demand that has two options: bring the gas or LNG to gas demand or build demand near the gas supply. The Government, through the Minister of Energy and Mineral Resources Decree (MEMRD) Number 2700.K/11/MEM/2012, has stipulated the Master Plan for the 2012-2025 National Gas Transmission and Distribution Network.
The Indonesian natural gas concession
The Indonesian natural gas concession chain can be divided into five groups of activities [9], namely: 1. Suppliers. Consists of producers and commercial enterprises that import natural gas until 2018, Indonesia had not recorded natural gas import activities from other countries. 2. Processing business activities. The companies engaged in purifying, enhancing the quality and value-added of natural gas such as producing Gas Fuel, processed products, LPG, CNG, and LNG. 3. Transportation business activities. They have to transport natural gas from a source or a storage or processing place. 4. Storage business activities. It means receiving, collecting, storing, and releasing gas fuel and processed products such as LNG, CNG, and PLG. 5. Commercial business activities: activities of natural gas sellers who buy natural gas from gas suppliers, distribute, and sell to end consumers for obtaining profit.
Natural gas users and priority scale
The allocation and utilization of natural gas for domestic needs is carried out in the order of priority [10], as follows: 1. Supporting Government Programs: provision for transportation, households and small customers, 2. Increasing national oil and gas production, 3. Fertilizer industry, 4. Natural gas-based industry, 5. Provision of electricity, and 6. Industries that use natural gas as fuel This priority sequence does not indicate the ranking of the amount of natural gas allocation. However, that order is based on the urgency of domestic needs.
Pricing type in the gas sale and purchase agreement
The gas sale and purchase agreement (GSA) mainly contains the volume of natural gas delivery, the point of delivery, and the price of gas. The price of natural gas in GSA can be divided into four types, as shown in the following table: Table 1. Types of gas prices in the GSA, compiled by the author
Gas Price Type Application Fixed Price
• Buyers of the type of power generation, and industries; • Used in the short-term gas trading Regulated (under Baseline Price) It used in government programs such as city gas and BBG Escalation Prices Widely used for power generation a and few fertilizer plants b Formulated Indexed with ICP • Used for buyers in the upstream (oil lifting) and downstream (refinery) business • In specific industries such as oleochemicals, rubber gloves, ceramics, and glass b Indexed with Brent crude or Japanese Crude Cocktail (JCC) Commonly used for spot LNG exports or medium-term contracts Price is associated with the price of the Product Used for a small number of specific industries such as referring to the price of ammonia, urea, and steel a Before being implemented of MEMRD Number 90.K/10/MEM/2020 b Before being implemented of MEMRD Number 89.K/10/MEM/2020
Natural gas price component
In general, the Indonesian natural gas price can be divided into two groups, namely the Upstream Gas Price and the Downstream Gas Price. The downstream gas price is the gas price at the end-user after adding infrastructure management costs (transmission and distribution) and trade costs as in Figure1. The natural gas transaction is carried out at the point of delivery between the producer or supplier and the user. In the sale of natural gas through pipelines, the delivery point is generally located at the wellhead. However, it can be outside the wellhead, namely the dedicated upstream pipeline. This pipe is built and owned by the upstream oil and gas contractor, which is connected to the plant gate or pipes owned by consumers or transportation services. Meanwhile, there are two models for LNG sales from upstream oil and gas, namely Delivered Ex Ship (DES) and Free Onboard (FOB).
The determination of natural gas prices is carried out with three considerations, as in Table 2. The field economy is determined as the baseline for natural gas prices. It is used to ensure the sustainability of upstream oil and gas investment. The price for the Public Service Obligation is the price for the city gas program and BBG transportation, which is below the market price. The ceiling price is a consideration for the upper limit of natural gas prices for domestic. If domestic consumers cannot meet the economy in the field, the contractor can export.
Baseline Price
The economics of the upstream project Ceiling Price Domestic and international gas prices Added value from domestic use of natural gas The purchasing power of domestic consumers Prices of fuel or energy substitutes
Public Service Obligation
The support for government programs to supply natural gas for transportation and households and small customers
Indonesian natural gas price regulations
The Government issued regulations related to natural gas pricing. The Government tries to set gas prices would be more competitive to end consumers and could provide a multiplier effect than just a commodity. The reviews are carried out at various levels of laws and regulations governing natural gas pricing, which are currently still valid, among others.
Upstream prices of natural gas
In the cooperation contract scheme in the upstream oil and gas sector, whether in the form of Conventional PSC or Gross Split, the profit-sharing received by the Government and Contractors is very dependent on gross revenue. The amount of Gross Revenue is determined by the selling price of oil and gas. Thus, in Indonesia, the price of natural gas by pipeline or LNG must be stipulated by the Minister, after receiving consideration from SKK Migas. The economy strongly influences the price of upstream gas in the market as a baseline and the quality of natural gas as raw material and fuel. In the Kalimantan region, the low price of natural gas is due to the abundant reserves that help the economy in the field. In contrast, for the West Java region, the low gas price is due to the high content of impurities in the natural gas produced. The high gas On the producer side, sales of natural gas must reach a particular economy in the market, which depends on the MARR of each company. The investment cost determines the calculation, the number of reserves, the profit-sharing scheme, and the gross revenue from the sale of oil and natural gas. Then, on the buyer side, it will consider the average gas price in the surrounding location, the quality of natural gas, and the incurred transportation costs.
In simple, we present the upstream gas prices in Table 3 by referring to the applicable regulations. For some applications, the price of gas can be set below its economical prices, such as for government programs, electricity, and specific industries. We will discuss this in the next section. In the last five years, the price of natural gas for several users has increased significantly. This increment was because an increase in baseline price of natural gas due to new contracts, the extensions of the supply chain, and challenging economies. Moreover, the ICP price increase and contract price escalation affect the increase of upstream price.
Gas prices for government programs.
Currently, there are still no binding and standard rules. The upstream gas price for the city and the SPBG is set at 4.72 USD / MMBTU through the Ministerial Decree. It applies specifically to each of its regions does not apply to the entire program. However, the prices at all locations are set at the same price.
Gas prices for oil lifting.
The use of natural gas to increase national oil and natural gas production is defined as an effort to increase production in support of the national oil and gas availability. Its use in connection with production makes the gas price formulated with the Indonesian oil price.
Gas prices for certain industries.
Since the issuance of Presidential Decree Number 40 of 2016 regulates the price of natural gas for specific industries at 6 US$/MMBTU. Several gas sales and purchase contracts have followed this rule. In April 2020, the Minister of Energy and Mineral Resources issued a regulation as a legal basis for lowering gas prices for specific industries and electricity and repealed previous regulations and decrees. In the industrial sector, the Minister of Energy and Mineral Resources Regulation (MEMRR) Number 8 of 2020 is issued with implementation instructions in the MEMRD Number 89.K/10/MEM/2020.
Gas prices for electricity
The Government issues MEMRR Number 10 of 2020 and implementation instructions through the MEMRD No. 91.K/12/Mem/2020 of 2020. The price of natural gas for electricity is set at 6 US$/MMTU at the consumer plant gate, namely PLN and the Electricity Generating Business Entity (BUPTL). Gas price adjustments do not affect KKKS revenue because the reduction in revenue will be deducted from the Government's Share.
Gas prices for another industries
The determination of the natural gas price for industry takes into account the economic price, the surrounding natural gas price, and the added value of its utilization. Determination of buyers and gas prices can be done using a beauty contest method. The bidder with the best price and has collaborated with the infrastructure owner will have a more significant opportunity. This mechanism is used to ensure that the Government and contractors receive optimal benefits and more planned and effective utilization.
Gas flare prices
Utilization of Flare Gas can be carried out by Buyers, namely (a) Business Entities holding Processing Business Permits and Natural Gas Trading Business Permits; or (b) Government Institutions that own or control the infrastructure for the distribution or use of Flare Gas. For buyers from Government Institutions, the maximum is set at 0.35 US$/MMBTU, and no escalation, Take or Pay, also SBLC are applied.
Downstream gas price
The downstream natural gas price is divided into two components, namely, infrastructure management costs and trade costs. Infrastructure management costs include transportation through transmission, distribution pipelines, distribution of dedicated downstream pipes, natural gas liquefaction, compression, regasification, and storage and transportation of LNG/CNG. Trading costs represent commodity management costs, consumer management costs, marketing costs, risk costs, and trade margins. Apart from these two components, downstream business entities are also subject to business activity fees. Costing provisions can be seen in Table 5. [19] Other infrastructure management costs such as regasification and LNG/CNG transportation Reported to the Government with rational calculation [19] Trading Cost Commodity management, consumer management, marketing and risk costs, and trade margins A maximum of 7% of the purchase gas/LNG price If the distribution is through more than one commercial business entity, then it is shared among them. [20] Business The tariff of transporting natural gas by pipeline is regulated and determined by the Regulatory Agency (BPH Migas). The tariff determines the natural gas owners' interest, business entities holding oil and gas transportation business permits, and consumers of natural gas [22]. Service activities in the downstream natural gas sector are also subject to VAT of 10%. BPH Migas sets the selling price of natural gas for households and small customers. The selling price of natural gas for non-households and small customers is regulated and stipulated by the Minister following statutory provisions.
Conclusions
The price of Indonesian gas is highly dependent on the policy issued by the Government. This model was triggered by the upstream oil and gas production sharing contract scheme, which was influenced by the gross revenues of natural gas sales. For electricity and specific industries, starting from April 2020, more competitive rates will be set to support the economy. This price is below the economical price, and the difference in reduction is taken from the Government's share. In the use of natural gas for other sectors, the Government is more flexible in setting upstream natural gas prices except for the Government Programs. In the downstream sector, pricing for transportation, distribution, and trade fees is determined by the upper limit price. Meanwhile, for other infrastructure management, fair prices are used based on business entity reports. This regulation makes the natural gas infrastructure business rigid and challenging to develop. The increment of upstream gas prices occurred because of the increase in ICP, the increasingly tough field economy, and the escalation factor of the price in GSA. | 2021-06-29T20:02:32.370Z | 2021-01-01T00:00:00.000 | {
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247200246 | pes2o/s2orc | v3-fos-license | Literature Class Environments Rich in Texts Supporting the Excellent School Literature Movement
Text-rich, interesting, and accessible literacy for Special Education Needs (SEN). This study aims to describe the embodiment of the text-rich, attractive, and accessible literacy classroom environment in supporting the literacy movement in Special Education Needs Schools. A qualitative approach with a single holistic case study method review design is applied. The research procedure includes problem identification, objectives, research questions, literature review, case selection and limitation, data collection, data analysis, triangulation, and integration of findings. The results show that the school literacy environment is quite good, especially to support the school literacy movement focused on activities 15 minutes before learning; the text-rich, accessible, and engaging literacy classroom environment has not been realized and has not supported literacy learning, the role of teachers, literacy teams, school committees, and leadership is not optimal. There are complex problems in realizing a literate classroom environment in special schools, including financing, commitment, and creativity of school residents (literacy team, teachers, students, and parents/stakeholders).
INTRODUCTION
The realization of a literacy environment rich in supporting texts for the School Literacy Movement (GLS) in Special Schools (SLB) is a mandate from the Minister of Education and Culture Regulation Number 23 of 2015 that must be fulfilled. In addition, it is also for the development of student literacy competencies and teacher professionalism. Dyna (2012) stated that education that focuses on improving the quality of the literate classroom environment would support teacher professionalism in learning because text-rich literacy materials facilitate it. The goal is to be used throughout the day to improve the literacy of special education needs (SEN) students. Printed materials are a meaningful resource because they are easy to read and provide children with valuable experience developing early literacy skills. However, research findings [1] indicate that the quality of inclusive schools' structural literacy environment in Indonesia is, on average low to moderate. This condition is not much different from the literacy environment in Special Schools, maybe even lower, even though NES requires a literacy environment rich in text, interesting, and accessible for the success of NES learning with various characteristics and specificities. This requires applying universal learning design (UDL) principles, namely providing equal access to NES according to their conditions [2]. Supporting this, [3] [4] states that it is important to create a classroom environment that helps improve attention, reduce anxiety, support regulation of NES behavior and emotions with various needs (such as autism spectrum disorders, ADHD, anxiety disorders). disorders, learning disorders, and sensory processing disorders require a supportive environment to function well), but generally, such environments are often neglected. [5] recommends a good classroom environment, namely: bright lights, quality classroom air, comfortable temperature, organized classroom settings. In addition, it also considers the elements of the 21stcentury classroom environment [6] no. 87 of 2017 concerning PPK (love to read) through 15 minutes of reading non-lesson books before the day of learning [7].
In addition, to support literacy that must be mastered by all students, namely basic literacy, which includes six categories, namely: literacy, numeracy, scientific literacy, financial literacy, civic literacy, and digital literacy. So six basic literacy skills must be mastered [8]. NES even though their abilities are limited, especially intellectual disability, teachers still have to try to develop the abilities of all NES according to their potential. Recent developments on the GLS guidelines provide a more detailed analysis of literacy, including The SLB literacy movement aims to create a literacy climate that includes: a) the physical environment of the school (availability of facilities, literacy infrastructure); b) social and affective environment (support and active participation of all school members) in carrying out SLB literacy activities, and c) academic environment (there is a simple literacy program that all school members can implement) [8]. These three objectives are a unity to improve the literacy of children with special needs. The three are interrelated. In detail, it is also stated the need for special infrastructure facilities that need to be equipped for each type of ABK.
Literacy support facilities and infrastructure for each type of ABK are stated in [9], that infrastructure for visually impaired students includes original/miniature 3dimensional objects, Braille books, audio CDs, tape recorders, flash disks, reglets, writing paper Braille, talking computers, display boards embossed or equipped with Braille descriptions, reading corners or shelves containing books that are fun for students to read. Infrastructure facilities for the Deaf, including picture storybooks, audio-video CDs, picture textbooks, hearing aids (groups/individuals), tape recorders, writing paper, display boards, reading corners, or shelves containing books fun reading for the Deaf. The infrastructure for intellectual disability, physically disabled, and autistic children is the same, namely: original/miniature 3dimensional objects, picture storybooks, audio-video CDs, tape recorders, hearing aids (groups/individuals), tape recorders, computers/laptops, flash disks, writing paper, display boards, and reading corners or shelves containing fun reading books for intellectual disability, physically handicapped, and autism.
Based on this description, the purpose of this study is to describe the literacy environment of schools and classrooms that are rich in text, attractive and accessible to NES, (2) participation of school residents in creating a literacy environment, (3) implementation of the School Literacy Movement, (4) supporting factors and barriers to the realization of a literacy environment that supports GLS.
METHODS
A qualitative approach with a single holistic case study method [9] is used in research because it seeks to describe human life and actions specifically in certain locations by only focusing on one case with the main problem of realizing an accessible and attractive literacy environment for NES in supporting SLB school literacy movement. The case study design refers to the Erlinger review because the procedure is simpler: problem identification, objectives, research questions, literature review, case selection and limitation, data collection, data analysis, triangulation, and integration of findings. The research location is in Sidoarjo; the research informants consist of 3 literacy teams, five teachers, two school leaders, two school committee representatives, two parents, and five students.
By environmental observation instruments. The data were collected using techniques, direct observation and participatory observation, interviews, documentation, archival records, and physical devices with the principle of multi-source evidence, creating case study databases, and maintaining evidence sets. Qualitative data analysis procedures [9] include pattern matching, explanation construction, time series analysis, logic models, and cross-case synthesis-power validity and reliability through triangulation of methods, sources. Research instruments include observation instruments, documentation, interview archives about the school literacy environment, and interview class literacy environments according to ABK and level (TKLB, SDLB, SMPLB, and SMALB) instruments to enrich the observation data.
RESULTS AND DISCUSSION
The research problems identified in the introduction include the complexity of realizing a text-rich and accessible/friendly literacy classroom environment for NES, consisting of TKLB/SDLB/SMPLB/SMALB levels. School infrastructure facilities and literacy support classes, school community participation, and factors supporting and inhibiting the realization of a literacy environment are described as follows.
Rich and accessible School Literacy Environment and Classroom Environment for ABK
a rich and accessible school literacy environment for children with special needs, in the form of suggestions for public school infrastructure and facilities according to the type of needs of children with special needs (blind, deaf, mentally retarded, physically handicapped, and autistic) stored in the laboratory of each special child with special needs, two school libraries for Kindergarten/SD level and the SMPLB/SMALB level. The literacy Advances in Social Science, Education and Humanities Research, volume 618 environment outside of school is quite good but needs to be developed again by adding basic literacy posters that teachers must develop and large display boards outside the classroom for each level (TKLB, SDLB, SMPLB, and SMALB). This display board serves to place students' work to motivate them to work individually and involve parents. In addition, it also shows the life of the school literacy movement at SLB Gedangan.
General School Literacy Support Facilities and Infrastructure. The school's facilities to support all activities are quite complete with fairly good facilities and support for the efforts of school leaders and staff for continuous improvement in developing the school. The infrastructure facilities in question include (1) 1 principal's room, (2) 1 administrative room, (3) 19 classrooms, (4) 1 computer laboratory room, (5) 1 screen printing workshop room, (6) 1 room automotive workshop, (7) 1 catering skills room, (8) 1 multimedia laboratory room, (9) 1 BPBI room (Sound and Rhythm Perception Development), (10) 1 self-development room (11) 1 music room, (12) 1 hall room, (13) 1 library room, (14) 1 sewing skills room, (15) 1 prayer room, and (16) physical therapy room, and (17) 2 student bathrooms and one teacher bathroom. The school facilities are quite complete and support all school activities and support activities for the development of school literacyespecially the multimedia laboratory, library, BPBI, selfdevelopment room, computer lab. The utilization of this infrastructure needs to be maximized with functional programs for ABK.
The school library has two rooms available for the TKLB/SDLB level; one room is located at the SDLB Tunagrahita location and is provided for the SMPLB/SMALB level. This is for space-saving and limited personnel, limited space conditions due to the construction of classrooms, and building repairs. The condition of the library is quite good. However, improvements are still needed in (1) library staff, (2) setting a comfortable library environment, (3) administration, (4) adding new book collections that suit the needs of each level and type of ABK, (5) scheduling activities in the library, and (6) activities to motivate students to be happy in the library.
Classroom literacy environment and Class Facilities. There are currently 19 classrooms that are not ideal enough to meet all needs according to the school level, class level, and type of ABK. Ideally, TKLB 2 classes, SDLB 6 classes, SMPLB 3 classes, and SMALB 3 for each type of disorder. Meanwhile, the class is divided for the distribution of students according to this education database (Dapodik). The construction and addition of classes continue to be pursued.
Regarding the literacy classroom environment, teachers and school residents have not understood and have not developed it. Conventional classroom facilities and their arrangement are not by the needs of students who are accessible to make it easier to move and be creative. Large and heavy inflexible tables and chairs are not easy to move and not accessible to crew members, narrow, and not free for their activities. Reading corners, display boards, an environment rich in writing with various media types to enrich the class is not yet visible, for example, word walls, cards, big books, pop-ups, a dictionary of books suitable for the student level. Therefore, a classroom surgery model is needed to motivate teachers to be creative in arranging classes in a literacy-rich environment.
Reading Corner. A reading corner to support literacy activities has been attempted, especially a reading corner for activities 20 minutes before the start of the lesson, which is held on Tuesdays and Wednesdays with variations in implementation adapted to NES conditions. There are 6 reading corner blocks for students in the activity block 20 minutes before the lesson starts according to the student's condition and abnormalities (deafblind block, deaf block; SD block, blind block, SMPLB block, and high school block). The reading corner in each block has not yet been displayed; the books need to be displayed; for this reason, it is necessary to choose books that are by each school level and the level of the book level. For the safety of books, considering the condition of students who vary in their behavior and concerns about behavior that deviates from the reading corner, it is necessary to think about the safety for students and the safety of the book itself. The reading corner is designed in the form of a tube for a bookshelf. Unfortunately, this reading corner has not been filled with books to motivate students' interest in reading and enjoyment. The open reading corner shelf design does not allow for permanent book arrangement.
The Guardian/Guest Reading Area is available at the gate. The reading corner for parents/guests is located in the north corner of the gate. There is seating for the waiting room for parents and student companions. This waiting room can be used by anyone who needs it with the school as a waiting room.
There is no display board outside the school yet. It is necessary to design this display board for the degree of work of students at each level (TKLB, SDLB, SMPLB, SMALB). In this case, the arrangement for the type of ABK needs to be considered; strategic placement locations are affordable and easily accessible to all students to be directly involved in the arrangement. The most important thing is the consideration of student safety and infrastructure.
Special ABK Infrastructure Facilities. Special facilities for special needs children with special needs are quite complete, centralized in special laboratories including BPBI room for language development for Deaf Children, Blind Laboratory, Self-help laboratory for independent development of mentally disabled children, physical therapy room for physically disabled children,
Advances in Social Science, Education and Humanities Research, volume 618
computer laboratory, multimedia laboratory, music room and vocational laboratory (culinary, automotive, and sewing). All of them support GLS activities. For this reason, the effort to label each existing material is important to maximize infrastructure in supporting the development of student literacy. Special infrastructure in the classroom also needs to be developed by the teacher with simple media or equipment. The involvement of parents is important to create an atmosphere of literacy in the classroom.
Participation of School Citizens in Creating a Literacy Environment
The Principal/Principal and Deputy Principal's participation is enthusiastic about developing schools with various programs and school construction. The development of the school iteration environment is not yet major, although it shows enthusiasm and enthusiasm in its development but is not optimal, active as a literacy activist as an organizer of SLB literacy competitions. The encouragement for the literacy team is very good but is constrained by the cost factor.
The participation of the Literacy Team is sufficient and moderate; there is a public school environment, in the form of banners, reading corners outside the classroom, for each specialty and level (TKLB/SDLB/SMPLB/SMALB) and a reading corner for guests and guardians of students. Libraries for each school level but not well managed, literacy parks are also evidence of the creativity of the literacy team. The ups and downs of the spirit of developing literacy by the team.
Teacher Participation. It is classified as low and moderate; some teachers are not paying attention to their class, the iteration environment is limited to classroom facilities, desks, chairs, blackboards, writing tools. Reading corner, pictures are not yet available. Teachers do not yet understand the nature of developing a literacy environment in the classroom and schools to successfully implement GLS in special schools.
School Committee participation is low because it has not been fully empowered. They have taken advantage of the reading corner but have not yet organized the literacy classroom environment or the literacy school environment.
Implementation of the School Literacy Movement
The school literacy movement is understood as an activity 15 minutes before learning begins. At SLB Gedangan, the activity was carried out for 20 minutes. The activity begins at 06.30, preparation with the accompaniment of songs that students and teachers understand as the beginning of the activity. Instructions from the microphone invite students to gather. The teacher reads for students who cannot read; students do not read alone and do not bring books. For students who have been able to read, they are reading independently. The teacher's direction is carried out by asking questions. This activity is routine on Tuesdays and Fridays.
Supporting and Inhibiting Factors in the realization of a Literacy Environment GLS supporter
The supporting factor for the realization of a literacy environment is the carrying capacity of school leaders who have strong motivation and enthusiasm and the ability to cooperate with the Surabaya Education Office and are often involved in the Ministry of Education and Culture activities. Leaders are young and high-spirited. The literacy team is a good support force that always encourages the development of the literacy environment. The school committee, if empowered, is strong support. The school is wide and is often used as an arena for competitions in a local pilot school.
The inhibiting factor is the teacher's lack of understanding of the school literacy movement. Many teachers are old-lack of opportunities in teacher training, minimal funding.
CONCLUSION
The school literacy environment, in general, is quite good with the existing infrastructure, quite accessible but not adequate. The literacy team has attempted to provide literacy facilities according to the demands of the GLS-SLB by utilizing the existing media and infrastructure even though they are quite old. As well as trying to add new infrastructure, but very limited. Current technological advances need new infrastructure facilities. In general, the infrastructure for each class has not been managed. The narrow space is compartmentalized (2m x 2m). School participation is still low. The carrying capacity is quite adequate-barriers, especially funding and low school community participation.
SUGGESTION
The development of a literacy environment needs to be carried out by referring to the guidelines and theories of a literacy-rich environment. The school library competition that Elementary Schools have carried out should be imitated. Conventional classroom infrastructure with large tables and chairs for NES is inflexible and inaccessible and needs to be replaced gradually by submitting to a better PKLK. The collaboration of the Community Service Team in Higher Education with the Education Office, PKLK, and Quality Assurance and schools is very extraordinary. | 2022-03-03T16:30:27.986Z | 2022-01-01T00:00:00.000 | {
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226872741 | pes2o/s2orc | v3-fos-license | UNRESOLVED TACHYPNOEA: A PRESENTATION OF CONGENITAL COR-TRIATRIATUM
Cor-triatriatum is a rare cardiac anomaly. In literature, majority case reports on the condition focused on its late presentation in adulthood. It can be easily corrected by surgical intervention to avoid pulmonary congestion and subsequent pulmonary hypertension. We report a rare case of cor-triatriatum with severe pulmonary hypertension in a 7-week-old baby who presented with persistent tachypnoea.
Introduction
Cor-triatriatum is a rare cardiac malformation and may present with variation in clinical presentation. Its incidence is estimated at about 0.1% of all congenital heart disease. In this anomaly, the atrium is divided by a fibromuscular membrane into two distinct chambers: a posterior-superior chamber which receives the four pulmonary veins and an anterior-inferior chamber (true left atrium) which connected to the ventricle via atrioventricular valves. Normally, there is a small opening connecting these two chambers. There are two types: cor-triatriatum sinister and cor-triatriatum dexter for left and right atrium respectively and the later was extremely rare. We reported a case of cortriatriatum sinister in a 7-week-old baby who presented with respiratory symptoms.
Case report Baby X was born normally at full term with the birth weight of 3.3 kg. He presented with a short (1 week) history of tachypnoea and interrupted feeding. He was commenced on the intravenous antibiotic for suspected lower respiratory tract infection despite negative infective markers. His respiratory rate was 70 breaths/minute at the initial presentation.
Following completion of antibiotic treatment, baby X maintained his respiratory rate at 65 breaths/minute with saturation of 97% in FiO2 0.35. There was no adventitious breath sound on auscultation, apex beat was palpable at the fourth intercostal space midclavicular line with the presence of loud second heart sound at the pulmonary area. His liver edge was palpable 1 cm below the right costal margin. His chest radiograph showed normal cardiac contour with the cardio-thoracic ratio of 0.51. There was haziness of the left upper zone with prominence pulmonary vascular markings. Echocardiography revealed situs solitus, levocardia with dilated right atrium (RA) and right ventricle (RV), presence of patent foramen ovale (PFO) size measured at 5.9 mm with bidirectional flow which connected between the upper chamber of the left atrium and right atrium, moderate to severe tricuspid regurgitation [TR] with pulmonary gradient (PG) of 80 mmHg with velocity of 4.7m/sec, dilated main pulmonary artery at 13 mm, aortic valve annulus of 9.2 mm and pulmonary venous (PV) annulus of 11.8 mm. Fibromuscular septum dividing left atrium (LA) into the upper and lower chamber connected through a small orifice measuring 4.8 mm was seen.
He was subsequently transferred to the cardiac centre for surgical correction. The large PDA was found intraoperatively, which was ligated successfully. There was a stormy post-operatively period where he required a delay chest closure, complicated with pulmonary hypertension crisis requiring inotropic support and inhaled nitric oxide. He was discharged home on day 10 postoperatively. At 6 weeks follow up, no further evidence of pulmonary hypertension was noted on the echocardiogram. He was clinically well and gaining weight.
Discussion
Cor-triatriatum is a rare cardiac anomaly with a prevalence of <0.1% of all congenital heart disease and has a ratio of male to female of 1.5:1 [1]. It can be corrected easily by surgical intervention. Clinical and radiological signs are typical of those with pulmonary arterial and venous hypertension [2]. In cor-triatriatum sinister, the left atrium is divided by a fibromuscular membrane into two distinct chambers: a posterior -superior chamber receiving the four pulmonary veins and an anterior -inferior chamber that connects to the left ventricle by means of the mitral valve. Other associated anomalies are the unrooted coronary sinus, ventricular septal defect, coarctation of the aorta, atrioventricular septal defect, tetralogy of Fallot and rarely asplenia and polysplenia. Failure of incorporation of the common pulmonary vein into the left atrium lead to symptoms associated to pulmonary venous obstruction and pressure loading of the right side of the heart [3]. Faltering growth, recurrent pneumonia, feeding difficulties, tachypnoea, heart failure, cyanosis and arrhythmias are some of the reported presenting features.
There are various embryologic hypotheses to explain the development of cor-triatriatum -the entrapment and impingement theories [4]. Entrapment hypothesis postulated that the left horn of the sinus venosus entraps the common pulmonary vein and thereby prevents its incorporation into the left atrium. In the impingement theory, the persistent left superior vena cava on the left atrium induces the production of an abnormal left atrial membrane [5]. Often cor-triatriatum presents late in adulthood due to fibrosis and calcification of the orifice in the anomalous septum, the development of mitral regurgitation, or atrial fibrillation.
Diagnostic accuracy should be achieved using transthoracic or transoesophageal echocardiography and cardiac catheterization. Correcting cor-triatriatum should include finding its associated anomalies and delineating the complex congenital heart condition [6].
In our case, the patient has the anomaly in his left atrium thus is called cor-triatriatum sinister ( Figure 1) with type IIA1 based on Loeffler classification [8] (Table 1).
His tachypnoea persisted without improvement after standard antimicrobial therapy. There was no evidence of heart failure on the chest radiograph apart from plethoric lung field and normal cardiothoracic ratio (Figure 2). Besides pulmonary venous congestion features in cor-triatriatum, the large PDA was the factor that contributed to the development of the severe pulmonary hypertension. The PDA finding was missed on the initial echocardiography due to reversal shunt of the flow which sometimes was difficult to appreciate on echocardiography. The learning point here is -if the patient has unresolved tachypnoea, cardiac aetiology should be considered. Our patient had a successful surgical correction of cor-triatriatum, ligation of patent ductus arteriosus and closure of the patent foramen ovale at the tertiary cardiac centre.
Conclusion
Cor-triatriatum should be considered as differential diagnosis in a baby presented with unresolved tachypnoea in general practice setting, following the exclusion of respiratory aetiology. Despite its rare condition, early identification and diagnosis would result in a good outcome for the patient. | 2020-11-14T18:08:31.057Z | 2020-04-18T00:00:00.000 | {
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14652748 | pes2o/s2orc | v3-fos-license | Effect of cyclosporin A on proteinuria in the course of glomerulopathy associated with WT1 mutations
Denys–Drash syndrome (DDS) is characterized by progressive glomerulopathy caused by diffuse mesangial sclerosis (DMS), genitourinary defects, and a higher risk of developing Wilms’ tumor. It is commonly assumed that the DMS is unresponsive to any medications. In this report, we present a patient with Denys–Drash syndrome, in whom the cyclosporine A (CsA) was found to induce total remission. This observation and observations of other authors confirm that in genetic forms of nephrotic syndrome, the proteinuric effect of CsA may be due to a non-immunologic mechanism. We confirm the beneficial effect of CsA treatment in DDS; however, the potential nephrotoxicity of this drug will probably not allow long-term use.
Introduction
Nephrotic syndrome in the first year of life in two thirds of cases is caused by mutations in four genes (NPHS1, NPHS2, WT1, and LAMB2) [10]. The Wilms' tumor suppressor gene 1 (WT1) encodes a transcription factor involved in kidney and gonadal development [19]. Heterozygous de novo mutations in WT1 gene cause Denys-Drash syndrome (DDS) and Frasier syndrome (FS), two syndromes charac-terized by nephrotic syndrome (NS) with either diffuse mesangial sclerosis (DMS) in DDS or focal segmental glomerulosclerosis (FSGS) in FS, genitourinary defects and a higher risk of developing Wilms' tumor in DDS, or gonadal dysgerminoma in both DDS and FS [13].
Gellermann et al. [8] recently published three children with FSGS on renal biopsy and WT1 mutations, who responded to intensified therapy consisting of oral cyclosporine A (CsA) in combination with oral or intravenous glucocorticoids. CsA has been used in the treatment of idiopathic syndrome for 20 years [14]. The mechanism of CsA action in minimal change disease was the inhibition of nuclear factor of activated T cell (NFAT) signaling in T lymphocytes [15]. However, the therapeutic effect of CsA in genetic nephrotic syndrome is probably related to a direct influence of CsA on podocytes.
In this report, we present a patient with Denys-Drash syndrome, in whom the calcineurin inhibitors were found to induce total remission.
Case report
Our patient is the first child of non-consanguineous young parents (mother 32, father 34 years) without any past medical history of kidney diseases. The pregnancy was uneventful. The infant was born at 41 weeks' gestation (birth weight 3,450 g, length 55 cm). The delivery and neonatal period were normal. At the age of 7 months, she was hospitalized for the first time in the Pediatric Nephrology Department because of proteinuria and hematuria, which was found in routine urinalysis performed before the vaccination.
On admission, the girl was in good general condition. Physical examination revealed no edema, normal female external genitalia, and normal body weight and height for chronological age. Blood pressure was 93/49 mmHg and pulse was 120 beats per minute.
The serum creatinine level was 0.23 mg/dl, urea was 8 mg/dl, and the glomerular filtration rate was 133 ml/min/ 1.73 m 2 by the Schwartz formula. Antinuclear antibodies were negative, and the level of C3 and C4 were within normal limits. Congenital nephrotic syndrome was suspected. The ultrasound of the kidneys showed inhomogeneous parenchymal hyperechogenicity. Any image consistent with renal tumor was not seen.
Chromosomal analysis demonstrated karyotype 46, XX, and the biopsy of the kidney demonstrated diffuse mesangial proliferation and diffuse fibrillar increase, what may represent early stage of diffuse mesangial sclerosis. The glomerular lesions contained high amount of fibrils in the mesangial matrix with diffuse mesangial proliferation. The capillary walls were covered with hypertrophied podocytes. The glomerular basement membranes were thickened. A few tubules showed microcystic dilatation. Few pseudotubular structures in endothelial cells were found. Immunofluorescence on frozen sections using rabbit polyclonal antisera against human IgG, IgA, IgM, and C3 revealed only weak segmental IgM staining.
The molecular genetic analysis was performed in the laboratory at the University of Michigan in the USA. Mutation analysis of exons 8 to 9 of WT1 was performed, and the mutation R394W (c. 1180 C>T) was found in our patient. No WT1 gene mutations were found in the parents of the affected child.
The treatment with enalapril (1.25 mg/day), which was started after the admission and was continued for 11 months, did not change the proteinuria significantly. There was no reduction in proteinuria after 4-week oral prednisone treatment (60 mg/m 2 /day). Seven months later, we started the therapy with CsA (6 mg/kg/24 h in two divided doses). After 2 months of CsA treatment, partial remission was observed. The serum albumin level rose and proteinuria declined. The pre-dose/trough (C0) CsA level was 104.4 ng/ml. After 6 months of CsA treatment, complete remission was achieved. There was no proteinuria. The serum total protein was (6.47 mg/dl), albumin (4.26 mg/dl), cholesterol 197 mg/dl, and platelet count 385×10 3 /ql. Creatinine level was 0.32 mg/dl and the glomerular filtration rate was 147 ml/min/1.73 m 2 . The pre-dose/trough (C0) CsA level was 95.03 ng/ml. At the 6 month of the treatment, we decided for CsA dose reduction to 4.5 mg/kg, but a month later, slight proteinuria was observed. Now CsA treatment is continued with the higher dose (6 mg/kg/24 h). The patient is under the regular control of The Pediatric Nephrology Department. She has no proteinuria. Serum creatinine level is normal. The ultrasonography examination, performed every 3 months, has not revealed any signs of tumor. A control kidney biopsy is planned soon.
Discussion
We report the benefits of CsA treatment of the patient with glomerulopathy associated with WT1 mutation as seen in Denys-Drash syndrome. The girl in this report had earlyonset SRNS secondary to DMS, what was confirmed in kidney biopsy. Genetic studies showed mutations in intron 9 of WT1 gene c. 1180 C>T. This is the most common mutation found in DDS [8]. Mutations in exons 8 and 9 of the WT1 gene have been found in patients with isolated SRNS and in SRNS associated with Wilms' tumor or urogenital malformations. The data presented by Mucha et al. [16] indicated that screening of WT1 exons 8 and 9 in patients with sporadic SRNS is sufficient to detect pathogenic WT1 mutations. Our patient lacked any evidence of anomaly of the kidney or urogenital tract. No renal tumor has not been seen so far. It is possible that our patients have an incomplete form of DDS; however, it corresponds to the observation of Ismaili et al. [11], who reported the presence of Wilms' tumor in only one of two patients with DDS. Chernin et al. [5] in the recent publication suggested that missense mutations can occur with and without Wilms' tumor.
In our patient, the diagnosis of DDS was done in the eighth month of life. The girl fulfilled the criteria of SRNS. In this situation, CsA therapy was started with a significant reduction of proteinuria and remission of nephrotic syndrome. Three months later, complete remission of proteinuria was found.
Whereas genetic forms of nephrotic syndrome do not respond to therapy with immunosuppressive drugs, partial remission has been reported following therapy with calcineurin inhibitors in patients with NPHS2 [12], Frasier syndrome [8], mutations of the phospholipase C epsilon gene (PLCE1) [9], and Alport syndrome [3,4]. Gellermann et al. [8] reported three children with FSGS associated with WT1 mutation treated with combined CsA and prednisone therapy given for 6 and 12 months. In long-term observation, the authors observed remission of NS and significant reduction of proteinuria. Our patient was treated with CsA alone and we observed complete remission of proteinuria. This is interesting because it was considered that DMS was unresponsive to any medications [20].
The therapy does not seem to be accompanied by a significant loss of renal function on the short term. We plan to continue the treatment with the lowest possible dose which controls the proteinuria. A control kidney biopsy to check for CsA nephrotoxicity will be done soon.
Calcineurin is a ubiquitously expressed serine/threonine phosphatase [1]. It was considered that the main effect of CsA treatment in nephrotic syndrome caused by T cell dysfunction was the inhibition of NFAT signaling in T cells [6]. However, it was found that CsA can also reduce proteinuria in non-immunological diseases, raising doubts about the above hypothesis. In genetic diseases, mechanisms other than the immunosuppressive effects are involved in reduction of proteinuria. Similarly, the afferent arteriole vasoconstriction, which is induced by CsA, does not seem to play the main role in the reduction of proteinuria. This fact was suggested by Zietse et al. [21], who studied the influence of CsA on proteinuria in different glomerulopathies and found the reduction of protein excretion in minimal change disease, but not in membranoproliferative glomerulonephritis or FSGS. The possible mechanism in genetic NS may be that proposed by Faul et al. [7]. The authors suggested the influence of CsA on synaptopodin, an actin binding protein that was highly expressed in podocytes. It is also an important regulator of podocyte function through its interaction with CD2AP [2]. It was shown that calcineurin caused the dephosphorylation of synaptopodin and caused proteinuria via its degradation. In this situation, CsA therapy leads to the stabilization of its actin cytoskeleton. This mechanism seems to be possible because significant reduction (30%) of CsA dose caused the slight proteinuria in our patient, so we increased the dose to initial one. Our observation seems to confirm the theory of Mundel and Reiser, who suggested that proteinuria is due to an enzymatic disease of the podocytes [18]. It is likely that podocytes are a direct target of CsA, which preserves the phosphorylation-dependent synaptopodin-14-3-3beta interaction, protects synaptopodin from degradation, and preserves a stable filtration barrier [7,17].
The fact that the antiproteinuric effect of CsA results from a direct effect on podocytes explains not only its beneficial role in most steroid-resistant and steroid-dependent nephrotic syndromes but also the fact of cyclosporine dependency. Our observation and observations of other authors [3,4,9,12,15] confirm the beneficial effect of CsA treatment in genetic NS; however, the potential nephrotoxicity of this drug will probably not allow the long-term use. | 2014-10-01T00:00:00.000Z | 2010-09-17T00:00:00.000 | {
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17315656 | pes2o/s2orc | v3-fos-license | Systems Biology in the Context of Big Data and Networks
Science is going through two rapidly changing phenomena: one is the increasing capabilities of the computers and software tools from terabytes to petabytes and beyond, and the other is the advancement in high-throughput molecular biology producing piles of data related to genomes, transcriptomes, proteomes, metabolomes, interactomes, and so on. Biology has become a data intensive science and as a consequence biology and computer science have become complementary to each other bridged by other branches of science such as statistics, mathematics, physics, and chemistry. The combination of versatile knowledge has caused the advent of big-data biology, network biology, and other new branches of biology. Network biology for instance facilitates the system-level understanding of the cell or cellular components and subprocesses. It is often also referred to as systems biology. The purpose of this field is to understand organisms or cells as a whole at various levels of functions and mechanisms. Systems biology is now facing the challenges of analyzing big molecular biological data and huge biological networks. This review gives an overview of the progress in big-data biology, and data handling and also introduces some applications of networks and multivariate analysis in systems biology.
Introduction
Biology has recently become a "big-data science" mainly supported by the advances in high-throughput experimental technologies. Data-intensive science consists of three basic activities: capture, curation, and analysis [1]. All three of these phases of handling big data raise many new research challenges to pursue in systems biology. The big data challenges are not only their size but also their increasing complexity. The emergence of big data biological sciences, such as systems biology, and their growing impact on health, nutrition, ecosystems, and other societal issues have only recently become the focus of scholars in social studies, science, and information studies [2]. Jim Gray proposed the fourth data paradigm and farming of the "data deluge;" that is, the capacity to measure, store, analyze, and visualize data is the new reality to which science must adapt. The heart of the fourth paradigm is data and it sits alongside empiricism (1st paradigm), theory (2nd paradigm), and simulation (3rd paradigm), which together form the continuum we think of as the modern scientific method [1]. Systems biology is one of the several other subjects including astronomy, ecology, and meteorology where challenges of the fourth data paradigm have become relevant. The basic purpose of systems biology is the system-level understanding of a cell or an organism, which can be summarized in the context of molecular networks as (1) an understanding of the structure of all the components of a cell/organism up to molecular level, (2) the ability to predict the future state of the cell/organism under a normal environment, (3) the ability to predict the output responses for a given input stimulus, and (4) the ability to estimate the changes in system behavior upon perturbation of the components or the environment. In a cell or organism the primary-level components, for example, the molecules, are of numerous types and numbers and hence systemlevel understanding of a cell/organism is still a very difficult task. However along the way to achieve the theoretical goal of systems biology, that is, to understand life scientifically, many other practical applications will be invented. Practical applications will include development of new generation medical tests, drugs, foods, fuel, materials, sensors, and other applications. Systems biology now faces the challenges of analyzing large amounts of molecular biological data and huge biological networks.
Big Picture of Hierarchy and Networks in Systems Biology
The hierarchy shown in Figure 1(a) summarizes the major types of molecules being studied in systems biology, which aims to determine the functions of the molecules of each layer and how these molecules interact with each other within individual layers and between layers to perform biological tasks. Figure 1(b) shows the overview of the accumulated data in the KNApSAcK database which has been developed to facilitate the knowledge discovery regarding plants and plant-human omics [3]. The upper part of Figure 1(a) can be regarded as an example of a big picture of networks in systems biology. This figure implies the existence and abstraction of networks in individual species and across species. Numerous studies constructed suitable networks for understanding systems or subsystems within species. Networks representing systems or subsystems can also be compared or linked between species (Figure 1(b)). This world is cohabitated by humans and many other species and the understanding of the interactions at the molecular level among all the species is important for healthy and sustainable living for humans and other organisms.
Data Types in Systems Biology
Many experiments are conducted in systems biology like many other branches of science; these experiments produce various types of data. Currently in systems biology some of the popularly-used data types are as follows.
Sequences.
The DNA is a molecule of double helix structure that consisted of two complementary strands of sequences of four nucleotide bases-adenine, thymine, guanine, and cytosine, represented as A, T, G, and C, respectively [4]. DNA contains all the necessary information preserved in the order of the nucleotide sequences. Hence, it is important to determine the sequences accurately. A gene is usually a continuous part of one of the DNA strands and contains codes for one or a few different proteins. The proteins are essential molecules that consisted of amino acid sequences. From the starting site of a gene, every three nucleotides are called a codon and a codon corresponds to an amino acid. It is in this way that a gene preserves the code of a protein.
For example ATGAAGCTACTGTCTTCTATCGAACAA-GCATGCGAT is the sequence of the first 30 nucleotides of GAL4 gene of yeast and KLLSSIEQAC is the sequence of the first 10 amino acids of the corresponding protein. There is variation in codon usage by different organisms and links can be established between codon usage and the biological characteristics of an organism [5,6]. Development of DNA sequencing techniques started in the 1970s and since then various methods have been developed [7][8][9]. The sequence of individual genes, group of genes, parts of chromosomes, full chromosomes, or entire genomes are determined for different purposes. Recent developments in next generation sequencing techniques have greatly reduced the time and cost of sequencing [10][11][12].
Molecular
Structure. Determination and prediction of the three-dimensional structures of omics molecules are also very important. DNA is packed into protein-DNA structures referred to as chromatin mainly to fit the long DNA chain inside the cell. The primary protein components of chromatin are histones. The DNA packaging protects DNA from damage and plays important roles in gene regulation by allowing or blocking the binding of transcription factors and other molecules to DNA. Usually, proteins also work by forming complexes with other proteins. In general it can be stated that DNA, RNA, and protein molecules usually bind with one another dynamically to perform different cellular functions. Therefore, not only the sequences but also the threedimensional structures of the omics molecules are important for predicting the possibility of binding between molecules and thus predict the functions of uncharacterized molecules. X-ray crystallography, nuclear magnetic resonance (NMR), and electron microscopy are the experimental procedures used for determining the 3D structures of proteins. A number of methods for the computational prediction of protein structure from its sequence have been developed [13,14]. Also, there are computational methods for the prediction of RNA structures [15][16][17]. There are numerous software tools for predicting and visualizing 3D structures of proteins and RNAs. A comprehensive list of these tools can be found in scientific literature. Molecular structure data are therefore three-dimensional geometrical figures of versatile shapes or related information that can be easily converted to threedimensional structures usually with the aid of computer software.
Gene Expression.
Gene expression is the process of extracting information of a gene and is the initial step of producing gene products such as mRNAs which are usually translated to proteins and functional RNAs such as rRNA or tRNA. Gene expression is known to take place in all life forms, that is, eukaryotes (unicellular and multicellular), prokaryotes (bacteria and Archaea), and viruses-to generate the macromolecular machinery and building blocks for life. Though most cells in an organism contain the same genes, not all of the genes are used in each cell. Some genes are turned on, or "expressed, " when needed in particular types of cells. Microarray technology [18,19] allows us to look at many genes at once and determine which are expressed in a particular cell type and to what extent. Next generation sequencers are also currently used to determine the gene expression [20]. To say that "a gene is highly expressed" means many copies of mRNA corresponding to that gene are produced in the cell. The extent of expression of genes is usually measured for comparison by using samples collected under different experimental conditions, for example, sick and healthy tissues, normal cells or cells put under certain stress or starving. Gene expression data is usually represented as a matrix where the rows represent genes and the columns represent experimental conditions; that is, gene expression data are multivariate data.
Binding Sites and Domains.
Many important cell processes such as RNA transcription, DNA packing, DNA replication, DNA recombination, and DNA repair are initiated and regulated by binding of proteins to selected DNA sequences. A position weight matrix (PWM) is a commonly-used representation of motifs (patterns) in biological sequences [21]. A PWM, also called position-specific weight matrix (PSWM) or position-specific scoring matrix (PSSM), is a matrix of score values that gives a weighted match to any given substring of fixed length. A DNA-binding domain (DBD) of a protein is an independently folded domain that contains at least one motif that identifies and binds double-or single-stranded DNA. A DBD can recognize a specific DNA sequence usually known as a recognition sequence or have a general affinity to DNA [22]. The domains of proteins and the binding sites at DNA are therefore part of the sequences of the corresponding proteins and the DNA, respectively.
Protein-Protein Interaction (PPI). In cells, thousands
of different types of proteins act as enzymes-catalysts to chemical reactions of the metabolism, components of cellular machinery (e.g., ribosomes), regulators of gene expression, and so on. Some proteins play specific roles in special cellular compartments, whereas others move from one compartment to another carrying mass or information. A protein may work as an individual entity, but usually two or more proteins bind together and form a complex to carry out their biological functions. The RNA polymerase, a large molecular machine that copies information from DNA to produce mRNA, is indeed a big protein complex that consisted of many proteins. Proteins are generally bound together in a complex not by chemical bonds but by other forces. Usually PPI data are represented as binary relation between two proteins whether they are part of two-protein complex or multiprotein complex. All or a number of the PPIs of an organism can be represented as a network where a protein represents a node and an interaction represents an edge. Experiments that are used to determine PPIs are yeast two hybrid system (Y2H) [23,24], affinity purification coupled to MS (AP-MS) [25], and so forth.
Mass Spectrometry.
Mass spectrometry (MS) is an analytical technique that produces spectra (singular spectrum) of the masses of the molecules comprising a sample. The spectra are used to determine the elemental composition of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules, such as peptides, metabolites, and other chemical compounds. Mass spectrometry works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios [26]. Mass spectrometry data can be represented as 2-(molecular weight versus magnitude) or 3-(molecular weight versus magnitude versus time) dimensional arrays; that is, they can be treated as multivariate data.
Metabolic Pathways.
Living cells generate energy and produce building material for cell components and replenishing enzymes by the process of metabolism. All organisms live and grow by receiving food and nutrients from the environment. The foods are processed through thousands of reactions. In cells chemical reactions take place around-theclock, constantly breaking and making chemical molecules and transferring ions and electrons. These reactions are called metabolic pathways. All or a group of known metabolic reactions of an organism can be represented as a network where metabolites are considered as nodes and a reaction between them is represented as edges. The edges in metabolic pathways correspond to one or more enzymes. Metabolic reactions follow the laws of physics and chemistry and thus modeling of metabolic reactions requires considering many physicochemical constraints [27]. In summary, it can be said that in terms of structure, extensively-used data in systems biology consist of four types: sequence data, 3D-structure data, multivariate data, and network data. However, the present challenge is that the amount of data is expanding rapidly requiring new tools and algorithms for handling big data. One type of data can be converted to another type for convenience of analysis. In the following section we discuss how networks can be generated from multivariate data and sequence data.
Network Generation from Different Data Types
In multivariate data, entities are represented by multiple variables and each entity can be regarded as a point in a multidimensional space or as a profile wave sketched according to the data values. Therefore, to convert multivariate data to a network, it is necessary to use a metric or some kind of measure that can assess distance or similarity between two multivariate entities. Widely-used distance or similarity measures are Euclidean distance, Manhattan distance, Mahalanobis distance, Correlation, and so forth [28][29][30]. The value of correlation ranges from −1 to +1 and the higher the value between two multivariate entities the more similar the entities. The opposite of distance can be used as a measure of similarity. Usually similarity between each pair of entities is calculated and then a threshold similarity is decided based on statistical analysis or some other important criteria, for example, to ensure scale-free degree distribution of the generated network or something like that. After selecting the threshold, all entities of the multivariate data are considered as the nodes of a network and an edge is inserted between the pair of entities for which the similarity is more than the threshold. A weighted network can be constructed by considering the similarity values as the weights of the edges. Sometimes one type of network is converted to another type for the convenience of applying algorithms or for some other purposes. In [31] the metabolic pathways are converted to a simple network of enzymes/genes. After that, graph spectral clustering was applied to the converted networks corresponding to M. tuberculosis, M. leprae, and E. coli. It was observed that reactions belonging to fatty acid biosynthesis and the FAS-II cycle of the mycolic acid pathway in M. tuberculosis form distinct, tightly connected subclusters. Also, based on degree centrality and eigenvector centrality the important genes in the networks were determined and their functions were analyzed. In [32] a PPI network was converted to the corresponding line graph for the convenience of applying a clustering algorithm. The conversion to line graph helped to place the related proteins to densely connected regions or clusters and thus paved the way to obtain useful results by the application of a graph clustering algorithm.
Big Biological Databases
Curation and analysis become important after capturing data from various experiments. Curation includes storage, retrieval, spreading around the world, filtering and integrating the data. The engineering techniques for these jobs are already known, but when that data is in the petabyte scale, it becomes complicated. Algorithms and software tools developed for the analysis of biological data also face the problems of scalability when data becomes very big. However, many big databases have been created around the world for curation and analysis of biological data and their data volume and performance are gradually improving. DNA Data Bank of Japan [33] and GenBank [34] are big databases of primary nucleotide sequences of many organisms which are related to the bottom level (Genome) of the hierarchy shown in Figure 1(a). PGDBj is a portal website for the integration of plant genome-related databases [35]. Gene expression omnibus (GEO) from NCBI is a data repository of array-or sequence-based gene expression profiles. ATTED-II is a database of coexpressed genes [36]. Information about noncoding RNA (ncRNA) families and other structured RNA elements can be found in Rfam database [37]. For the sequences and annotations of microRNAs, a useful database is miRBase [38]. GEO, ATTED-II, Rfam, and miRBase are related to the transcriptome level of Figure 1(a). UniProt is a comprehensive and freely accessible database of protein sequences and functional information of proteins [39]. The PROSITE database [40] consists of entries describing the protein families, domains, and functional sites as well as amino acid patterns and profiles of them. BIND [41] and BioGRID [42] are databases of protein-protein interactions. UniProt, PROSITE, BIND, and BioGRID are related to the proteome level of Figure 1(a). A central archive of macromolecular structural data is wwPDB [43]. The data accumulated in wwPDB is freely and publicly available to the global community. There are four member sites of wwPDB as follows: RCSB PDB (USA), PDBe (Europe), PDBj (Japan), and BMRB (USA). NetPath [44] is a manually curated database of signal transduction pathways in human. For metabolic pathways KEGG is a rich and well known database. KNApSAcK is a metabolomics database which was initially developed as a species metabolite relational database [45] and afterwards extended to KNApSAcK family databases containing information about herbal medicines [46,47] and metabolite activities [3]. KEGG and KNApSAcK are mainly associated with metabolome level of Figure 1(a). A comprehensive list of the omics databases can be found by searching the internet with the term "list of biological databases. "
Multivariate Analysis in Systems Biology
After capture and curation of data, the next step is analysis. Algorithms for analyzing multivariate data developed for other applications are currently used extensively in systems biology. The well-known methods for handling multivariate data are related to dimension reduction, clustering, classification, and regression. Often, dimension reduction is done before applying other methods. Principal component analysis (PCA) is the popular algorithm for dimension reduction [48]. PCA is a mathematical process that converts the values of a set of possibly-correlated variables into a set of values of uncorrelated variables which are called principal components. This transformation assigns the largest possible variance to the first principal component and usually the sum of variance of first few components approaches the total variance of all the variables in the original data. Therefore, variable reduction is performed by replacing all the original variables by the first few components obtained from PCA analysis. Regression analysis is a process for estimating the relationships between dependent variables (response variable) and independent (predictor) variables. Most regression analysis techniques estimate coefficients to establish a linear relation between dependent and independent variables. Least squares regression [49] and partial least squares (PLS) regression [50] are popular regression techniques. In multivariate data analysis, classification is the problem of identifying the category of a new observation from among a set of categories. Support vector machine (SVM) is a popular algorithm for classifying multivariate entities into two categories [51]. A multivariate entity can be regarded as a point in a multidimensional space. Usually an optimum hyperplane is determined based on training data so that multivariate entities of one category fall on one side of the hyperplane, while the entities of the other category fall on the other side. The concept of SVM can be extended to classify multivariate entities into multiple categories. Another type of classifier is the neural network [52], which is a naïve way of electronically simulating the function of the human brain. It is difficult to make a single formal definition of all the methods considered neural networks in the scientific literature. Usually, a neural network consists of a layer of input nodes and a layer of output nodes and several hidden layers of nodes. A neural network can be trained to use it as a classifier of multivariate entities. A multivariate data vector can be applied to the input nodes and after mathematically processing values applied at the input nodes by functions associated to the hidden nodes some values are propagated to the output nodes, which are utilized to determine the class of the input multivariate entity. The functions associated to the hidden nodes are determined or optimized based on the training data. The naïve Bayes classifier [53] is another popular supervised classification technique applicable to multivariate data. This classification algorithm is named after Thomas Bayes (1702-1761), who proposed the Bayes theorem. However it is called naïve Bayes because it naively assumes that the features or variables that describe a multivariate entity are mutually independent. Naïve Bayes classifier usually computes the probability that 6 BioMed Research International a multivariate entity belongs to a certain class given its features. Usually a set of training data or well-defined probability density functions are used to estimate different probabilities required to classify a multivariate entity. Random forest [54] is another classification method. The random forest is an ensemble classifier which constructs multiple decision trees. Each tree is constructed using a subset of training data and a subset of variables. Class assignment is made by the number of votes from all of the trees. Random forests can also be used to rank the importance of the variables in a regression or classification problem. Some other classification algorithms are partial least squares discriminant analysis (PLS-DA) [55] and soft independent modeling of class analogy (SIMCA) [56].
Another multivariate technique common in systems biology is clustering. This is the task of dividing a set of entities or objects into several groups or clusters in such a way that the objects in the same cluster are more similar in some sense to each other than to those in other clusters. Clustering and classification are related concepts, but in the case of classification, the categories are known beforehand, whereas in case of clustering, usually the categories are understood after applying a clustering algorithm. Hierarchical clustering [57,58] is the widely used algorithm for clustering of multivariate data. Hierarchical clustering is subdivided into 2 types: agglomerative methods and divisive methods. Agglomerative methods proceed by a series of fusions of the objects into groups eventually encompassing all objects in a single group. On the other hand, the divisive method separates the objects successively into finer groupings, eventually keeping each object in a single group. Hierarchical clustering is a technique that organizes elements into a tree. K-mean clustering [59] and self-organizing mapping (SOM) [60,61] are also important clustering algorithms applicable to multivariate data. K-mean is one of the simplest unsupervised clustering methods. One disadvantage of Kmean clustering is that it is necessary to guess and set the number of clusters in the targeted dataset before applying the algorithm. In case of SOM, multidimensional data/input vectors are mapped onto a two-dimensional array of nodes. Data points assigned to a node or nearby nodes are considered as a cluster.
Data assimilation can be referred to as state estimation which is the process of combining a model with observational data to estimate the state of a system. By data assimilation, a quantity of interest is estimated by combining observational data with the underlying dynamical principles governing the system under investigation. There are applications of data assimilations in systems biology. The data assimilation technique was applied to elucidate the dynamics of time-lagged gene-to-metabolite networks of Escherichia coli [62]. State transitions in the transcriptome of Bacillus subtilis and in both transcriptome and metabolome of Arabidopsis thaliana were predicted using a data assimilation technique called linear dynamical system model [63].
Numerous researches have been conducted in systems biology based on multivariate data analysis. We briefly discuss a few examples below.
Application of BL-SOM.
Batch learning self-organizing map (BL-SOM) is a novel neural-network algorithm that has been applied to efficiently and comprehensively analyze codon usage in approximately 60,000 genes from 29 bacterial species simultaneously [61]. In the original SOM method [60], the initial weight vectors are set by random values, but in BL-SOM the vectors are initialized by PCA, which is a statistical method that performs linear mapping to extract optimal features from an input distribution in the mean squared error sense. This technique allows the resulting SOM to be independent of input vectors. BL-SOM makes it possible to cluster and visualize the genes of individual species separately at a much higher resolution than can be obtained with PCA because PCA works based on linear mechanism while SOM can be trained to adapt non-linear mechanisms. The organization of the SOM can be explained by the genome G + C and tRNA compositions of the individual species. This work further used SOM to examine codon usage heterogeneity in the E. coli O157 genome, which contains "O157-unique segments" (O-islands), and showed that SOM is a powerful tool for characterization of horizontally transferred genes. Another example of the application of BL-SOM is the investigation of the enzyme sequence diversity related to secondary metabolism [64]. Initially, a map was constructed by using a big data matrix that consisted of the frequencies of all possible dipeptides in the protein sequence segments of plants and bacteria. The enzyme sequence diversity of the secondary metabolic pathways was examined by identifying clusters of segments associated with certain enzyme groups in the resulting map. The extent of diversity of fifteen secondary metabolic enzyme groups was discussed. On the resulting map, six clusters were rich with fragments of monoterpene, sesquiterpene, diterpene, and triterpene synthases. Nine clusters are corresponding to eight types of phenylpropanoids which are flavonoid and isoflavonoid synthases. Five clusters were associated to acetyl-, O-methyl-, and N-methyl transferases. As a whole these results show sequence similarities between specific types of enzymes related to secondary metabolic pathways.
Application of PLS-DA Model.
PLS-DA is an example of a multivariate model that has been applied in systems biology a case study being our previous work on Indonesian herbal medicines, popularly known as Jamu. These medicines are prepared from a mixture of several plants. The plants are chosen so that the Jamu has the desired efficacy. Thus, the composition of the plants used in a Jamu formula determines its efficacy. A model using partial least square discriminant analysis (PLS-DA) has been developed to predict the efficacy of Jamu based on the information of plants used in Jamu formula [55]. In this analysis, among 3,138 Jamu medicines, the efficacies of 2,248 Jamu medicines (71.6) were correctly predicted. Hence, the efficacy in most Jamu medicines can be predicted on the basis of the ingredient medicinal plants. In addition, the regression coefficients of the PLS-DA model, which relates plant usage in Jamu as predictors and Jamu efficacy as response, can be helpful in determining which plants in the ingredients of Jamu are used as main ingredients, which contribute primarily to the medicines' efficacies, and which plants are used as supporting ingredients. Plants that act as main ingredients will have a significant effect on the developed model. Due to the absence of parametric testing for the PLS-DA coefficients, the evaluation for significance is performed using permutation testing, in which the distribution of coefficients under the null hypothesis is generated via resampling of the existing data. The resampling is performed by permuting the order of the responses (in this case, Jamu efficacies) while maintaining the order of the predictors (in this case, plant utilization as Jamu ingredients) so that the existing relationship between the predictors and the response is destroyed and a new data set is generated under the null hypothesis; that is, plant utilization in Jamu does not affect Jamu efficacy. If such resampling is performed many times and the PLS-DA model is applied on the new data generated from the resampling, the accumulation of the PLS-DA coefficients obtained from this process generates a distribution, against which a value can be calculated and subsequently evaluated for significance. From the testing, it was observed that 234 plants (
Network Analysis in Systems Biology
For system-level understanding, initially the elements of a system are connected based on their mutual relation and a network is formed. Global network properties such as average path length, clustering coefficient, and degree distribution [65] are determined to assess the overall characteristics of the network such as how they are formed, what model they fit, how robust they are, and how tightly the elements are connected. There are numerous algorithms for finding clusters in a network. As a flexible notion the densely connected regions of a network are called clusters. Also, there are precise definitions of network clusters such as k-core, k-plex, and n-clan [66][67][68]. In recent years network theory has been substantially applied in systems biology. Construction and analysis of biological networks have become highly popular among omics researchers. In the following section we discuss some of the applications of networks and network algorithms in systems biology.
Function Prediction.
Functions of many omics molecules or entities, for example, genes, mRNAs, proteins, and also metabolites, are still unknown. A system-level approach of predicting functions of an unknown entity is performed by constructing a network of that entity and other known and unknown entities. Usually, after constructing a network, some suitable clustering method is applied. There are versatile graph clustering methods such as based on density and periphery [69], random walk [70], betweenness centrality [71], and so on. Usually the entities belonging to the same cluster are considered to have similar function based on the hypothesis "guilt by association" and therefore if the majority of the members of a cluster have some known function, then the unknown members are also assumed to have that function.
Protein Complex Detection.
Protein molecules may act individually, but in most cases to perform a biological task they form complexes by binding with one or more other protein molecules. High throughput experiments such as yeast two hybrid system (Y2H) [23,24] and affinity purification coupled to MS (AP-MS) [25] are used to determine the global set of interacting protein pairs. Such protein pairs can be represented as a network which is known as a PPI network. Usually it is assumed that a set of proteins in a densely connected region in a PPI network correspond to a protein complex. A good number of researches have been conducted to computationally detect protein complexes by applying clustering algorithms to PPI networks [72][73][74][75][76]. In those studies it was shown that real protein complexes of yeast substantially matched with computationally detected protein complexes.
Prediction of Interaction.
The presence of statistically significant complementary domain pairs in interacting protein pairs determined in the context of a PPI network indicates that certain domains facilitate protein binding [77,78]. Thus the presence of complementary domains in two new proteins implies the possibility that they might interact inside the cell. Thus, PPI networks of one or more species can be used to first determine complementary domain pairs and then to predict interactions between new protein pairs corresponding to a species.
Analyzing Evolution.
PathBLAST [79] is a network alignment and search tool for comparing protein interaction networks across species to identify protein pathways and complexes that have been conserved by evolution. The basic method searches for high-scoring alignments between pairs of protein interaction paths, for which proteins of the first path are paired with putative orthologs occurring in the same order in the second path.
Information
Integration. Networks can be constructed by combining different types of information, thus being helpful for integrated analysis of different omics molecules based on their relations. An integrative network of C. elegans embryogenesis genes based on three types of data (proteinprotein interaction, expression profiling similarity, and phenotyping profiling similarity) was studied in [80]. This study showed that gene pairs connected by interactions supported by multiple data are more likely to belong to the same GO category. For example in [81] gene expression profiles and mass spectrometry profiles are merged by using appropriate normalization of the data and a combined network of genes and metabolites has been constructed which helped find related genes and metabolites. A very large network of more than 60,000 interactions was reported [82] by integrating transcription factor binding, PPI, and protein phosphorylation data of yeast. This network was found to contain 7 types of 3-molecule motifs involving kinases out of which 5 types were overrepresented. 8 BioMed Research International 7.6. Determination of Important Entities. It is easy to realize that in the context of a network all nodes are not equally important. For example, a node with very high degree is obviously more important compared to a node having degree 1 or 2. There is an important relation between vertex degree and functional importance of the vertices in biological networks [83]. It has been reported that in PPI networks the removal of highly connected proteins is more likely to have more lethal effect [84]. The importance of a node in a network is precisely and mathematically determined by the centrality measures, for example, degree centrality, closeness centrality, betweenness centrality, eigenvector centrality, and so forth. In [85] a list and definitions of 17 types of different centrality measures are presented. 7.7. Disease Diagnosis. Biological networks can be utilized to identify biomarkers for disease diagnosis. Even a subnetwork also might be a biomarker. Protein network and mRNA profiles can be integrated to identify subnetwork biomarkers, that is, highly connected genes of a subnetwork whose sum of expression can be a marker of a disease state. There are several network-based approaches for identifying disease genes and protein interaction subnetworks which are disease signatures [86][87][88]. The application of a network analysis to metabolic PET (positron emission tomography) data obtained from patients with Parkinson's disease resulted in the identification and validation of two distinct spatial covariance patterns associated with the motor and cognitive manifestations of the disease [89].
Drug Development.
Complicated diseases such as cancer, Alzheimer, mental disorder, and heart diseases are very complex and caused by multiple molecular abnormalities. The drug discovery process of these diseases needs to target not a single molecule but entire molecular pathways of various cellular omics networks. Recently biological networks, for example, PPI networks and gene expression networks, are extensively used to find drug targets [90][91][92]. In [93], a method for drug target identification was proposed by combining information about drug therapeutic similarity, chemical similarity, and protein-protein interaction network using linear regression.
Prediction of Drug-Drug Interactions.
Understanding drug-drug interaction is important for drug development and drug administration. A drug interaction is a situation in which a substance (usually a drug) affects the activity of another drug when both are administered together. Drugdrug interaction is a significant cause of adverse drug reaction, especially in population on multiple medications. Drugdrug interaction can be categorized into three types: pharmaceutical, pharmacokinetic (PK), and pharmacodynamic (PD). A prediction method of pharmacodynamic drug-drug interaction through protein-protein interaction networks is proposed in [94]. This work introduced a metric called "Sscore" that measures the strength of network connection between drug targets. Thus drug-drug interaction was determined by assessing the interaction between the drug targets in the context of the whole PPI network.
Comparison of Biological
Mechanisms. Different types of biological networks, for example, PPI networks, gene regulatory networks, and metabolic pathways, and so forth, are system-level representations of biological mechanisms. Interesting results were obtained by comparing biological networks with random networks of the same size [69,95] or biological networks derived under different contexts [96]. Usually such comparisons are performed in the context of global network properties like degree distribution, average path length, and clustering coefficient, and so forth. Though global level degree distribution of PPI networks of many species follows power law, subtle differences between PPI networks of different species can be found by using other concepts. Not only PPI network but also other types of biological networks of different species can be compared to decipher the differences in mechanisms to explain phenotypes and other useful matters. A distance measure called relative graphlet frequency distance is presented in [97] which is based on the frequency of undirected induced subgraphs of size three to five. This measure was used to compare PPI networks of E. coli and yeast with different artificial networks [98]. Another concept of comparing two networks especially regulatory networks is on the basis of network motifs which are reoccurring patterns in complex networks and thus in some sense similar to the motifs in gene or protein sequences. It is shown in [99] that three highly overrepresented network motifs are present in the transcriptional interaction network of E. coli.
Conclusions
To understand a living organism as a system we first need to understand a cell as a system. This means we need to comprehensively understand the functions of each gene/protein/metabolite and how they work as an individual or in a group. The advancement in molecular biological experiments is producing huge piles of data related to genome and RNA sequence, protein and metabolite abundance, protein-protein interaction, gene expression, and so on. It is important to handle these huge data efficiently and scientifically to understand the cell as a system and to develop new applications in biotechnology and biomedical fields. This, in turn, necessitates the usage of high speed computers and integrating knowledge from other branches of science, for example, statistics, mathematics, physics, chemistry, and so on. The data we need to handle is of old formats, but the present challenge is that it has grown very big and needs the integration of different data types. This can be done by developing efficient scaling techniques for the current software tools and statistical and mathematical models for data handling. The application of network theory and algorithms can facilitate analyzing and integrating big data. | 2016-05-12T22:15:10.714Z | 2014-05-27T00:00:00.000 | {
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267239687 | pes2o/s2orc | v3-fos-license | Mediation effect of hope on the relationship between inner strength and self-management in patients after percutaneous coronary intervention
Background Effective self-management can enhance a patient’s quality of life and delay disease progression. However, motivating patients to adhere to self-management behavior following percutaneous coronary intervention (PCI) remains a challenge. With the robust development of positive psychology and interdisciplinary research, the role of psychology factors in patients’ health behavior has increasingly garnered attention. This study, focusing on positive psychological qualities, aims to investigate the relationship between inner strength, hope, and self-management in patients post-PCI, and to analyze the mediating role of hope between inner strength and self-management. Methods A cross-sectional survey was conducted among 216 PCI patients from a tertiary hospital in Nanjing. Research instruments included a self-designed general information questionnaire, the Inner Strength Scale (ISS), the Herth Hope Index (HHI), and the Coronary Self-Management Scale (CSMS). T-test, analysis of variance, Pearson’s correlation analysis, and mediating effect test were utilized for statistical analysis. Results The average scores of the ISS, HHI, and CSMS were 81.46 ± 12.00, 35.94 ± 5.38, and 86.79 ± 14.84, respectively. Inner strength was positively correlated with hope and self-management (r = 0.867, r = 0.630, respectively; all P < 0.05), and hope was positively correlated with self-management (r = 0.671, P < 0.05). Moreover, hope had a complete mediating effect between inner strength and self-management (β = 0.630, P < 0.01). Conclusion The inner strength, hope, and self-management of patients with PCI are at a moderate level. Inner strength primarily influences patients’ self-management behavior through hope, suggesting that medical staff can target hope to help patients build confidence in life after illness, form and accumulate inner strength, thereby promoting their self-management and improving prognosis.
Introduction
According to the Global Burden of Disease (GBD) Study 2019, approximately 523 million people worldwide suffer from cardiovascular disease, resulting in 18.6 million deaths.Coronary heart disease (CHD) accounts for about half of these deaths (Roth et al., 2020).CHD, a prototypical cardiovascular disease, poses a serious threat to human health, with China also facing a CHD crisis (Liu et al., 2019;Zhao et al., 2019).As reported in 2021, there are 11.39 million cases of coronary heart disease in China, and the incidence is expected to continue to rise due to multiple risk factors such as aging (Writing Committee of the Report on Cardiovascular Health and Diseases in China, 2022).With the significant progress of interventional techniques, percutaneous coronary intervention (PCI) has become a crucial strategy for revascularization in patients with CHD (Gao, 2019).According to the Heart Disease and Stroke Statistics from the American Heart Association, inpatient PCI procedures ranged from 1,310,000 to 480,000 annually between 2006and 2016(Lloyd-Jones et al., 2010;Tsao et al., 2023).In Germany, about 4,000,000 PCI procedures per year between 2014 and 2017 (Huber et al., 2020).There were 751,728 PCI procedures in persons aged 30 years and above between 2000/01 and 2020/21 in Australia (Kumsa et al., 2023).The database of the National Health Commission indicates that as of 2022, the total number of PCIs in China reached 1,293,932, with a mortality rate of 0.37% (Pan, 2023).PCI can effectively alleviate ischemic symptoms and reduce patient mortality (Hoole and Bambrough, 2020).However, while PCI can save the lives of patients with CHD, it struggles to reverse the pathological state of vessels.Patients remain in a "survival with disease" status post-PCI, and the risk of cardiovascular events remains high (Freites et al., 2022).Furthermore, existing systematic review indicates that there is no significant improvement in the quality of life for patients after PCI (Hirao et al., 2023).
Relevant guidelines suggest that self-care is a specific measure for secondary prevention of CHD, beneficial in reducing the occurrence of cardiovascular endpoint events (Knuuti et al., 2020;Liu et al., 2023).The World Health Organization (WHO) emphasizes that fostering positive self-management behaviors represents the most effective way to enhance the quality of life for individuals with chronic illnesses (World Health Organization [WHO], 2002).However, it is challenging for individuals to adjust their lifestyle and adhere to self-management behaviors (Aggarwal et al., 2021).Therefore, improving self-management compliance has become a research hotspot in the field of coronary heart disease.
Orem believed that self-care is a series of self-regulating behaviors individuals undertake to maintain their growth, development, and the integrity and normal function of their own structure (Orem, 1981).Self-management after PCI consists a range of behaviors, including smoking cessation, adopting a balanced diet, engaging in physical exercise, managing sleep, psychological adjustment, adherence to prescribed medications, self-monitoring of recurrent myocardial infarction and timely intervention (Peterson et al., 2014).Previous studies have shown that higher levels of self-management are associated with higher levels of quality of life and health promotion behaviors, and lower levels of readmission (Cheng and Kotronoulas, 2020;Collins et al., 2021).This suggests that improving self-management ability is not only key to promoting a healthy lifestyle, but also an important strategy to save medical costs and optimize the allocation of medical resources.However, existing studies have shown that the self-management status of patients post-PCI is not optimistic, and motivating patients to adhere to self-management behavior remains a difficult problem (Sua et al., 2020;Zhu et al., 2022).Riegel et al. (2012) proposed a middle-range theory of self-care for chronic illness, defining self-care in the context of chronic illness as the process of maintaining health through the practice of health promotion and disease management.This theory identifies two underlying mechanisms in self-care for chronic illness: reflection and decision-making.Reflection helps patients understand and master the knowledge, skills, and principles of chronic illness management, while decision-making involves actions taken by patients after assessing the context of disease management and the coping resources available to them.Thus, effective self-care not only requires a grasp of health management knowledge but also demands sufficient behavioral motivation.Empirical findings draw similar conclusions that health education is a key component of behavior change, but general health education alone has been shown to be insufficient to motivate self-management in patients with CHD (Feldman and Sills, 2013;Peterson et al., 2014).
With the rise of positive psychology and the deepening of the "bio-psychological-social" modern medical model, the influence of internal potential on health behavior is gaining attention (Müller et al., 2022).Promoting patient behavior change through intrinsic potential and advantages may be a new approach to optimizing patient self-management after PCI.
Inner strength (IS), defined as a universal reaction where patients grow and gain strength from adversity, is considered a core resource for humans to overcome disease and promote health (Boman et al., 2015).In the early 2000s, Roux conducted a meta-integration of relevant qualitative studies and formed the middle-range Theory of Inner Strength (TIS) (Roux et al., 2002).The interpretive framework comprises four processes: (1) experiencing the pain brought by illness and finding meaning in life experiences (2) establishing connections with oneself, family, friends, and spiritual power (3) actively participating in the decision-making process; (4) taking proactive actions.Through the process of experiencing illness, individuals can gradually develop an inner strength.The TIS posits that by enhancing inner strength, individuals can better utilize internal and external resources, make informed choices and decisions, and effectively cope with challenging events, thereby improving self-management.Unlike the risk-and diseasebased medical service model, the TIS focuses on tapping into a patient's potential during illness, mobilizing intrinsic motivation for health promotion, and improving self-management (Viglund et al., 2013).However, most inner strength studies in the medical field focus on the elderly and tumor patients, with few focusing on CHD patients (Viglund et al., 2014;Boman et al., 2015;Smith et al., 2019).In terms of research methods, more qualitative studies are used to understand the experience of inner strength.Only Dingley showed that inner strength is an important predictor of self-management in female cancer patients through a cross-sectional study (Dingley and Roux, 2014).There is a lack of quantitative studies on inner strength in post-PCI patients, and limited information is available on the mechanisms through which the inner strength affects self-management (Dingley et al., 2001;Mendes et al., 2010).
Previous evidence has suggested that inner strength is a significant predictor of hope (Gottlieb, 2014).Patients find meaning in life, make new connections, and take positive action during their illness, thus feeling hopeful about the future.Research indicates that hope, as a positive psychological trait, is gradually receiving more attention in the management of chronic diseases.Snyder (2002) posits that hope is an internal cognitive assessment mechanism established by individuals to achieve desired goals.According to Snyder's Hope Theory, hope is a fusion of goaloriented thinking, pathway thinking, and agency thinking.Goal is the core component of hope theory, and the specific manifestations of individual actions depend on the goals set.Pathway thinking is the cognitive aspect of hope, representing practical skills derived from personal internal psychological planning.Through pathway thinking, individuals can identify paths and strategies to achieve their goals.Agency thinking initiates individual actions, serving as the motivational component of the theory that propels individuals along the designated path toward their goals and sustains their perseverance and resilience in overcoming challenges.Individuals with high levels of hope often have clearer goals, stronger path thinking, and dynamic thinking.They are more inclined to proactively adopt positive coping strategies to deal with problems, thus improving patients' self-management behavior (Snyder, 2002;Corn et al., 2020).Previous studies have demonstrated a positive correlation between hope and self-management (Veres et al., 2014;Zhang et al., 2023).However, evidence of hope in PCI patients in the Chinese context is lacking.Thus, the relationship among inner strength, hope, and self-management remains unclear.
Research specifically focused on inner strength among PCI patients, and the associated issues of hope and self-management, are lacking in mainland China.Therefore, one of the objectives of this study was to understand the status of self-management, inner strength, and hope in patients post-PCI.In addition, according to Fredrickson's Broaden-and-Build Theory of Positive Emotions, positive emotions, such as hope, play a crucial role in establishing enduring internal resources.These resources empower individuals to fully engage their initiative, fostering a range of thoughts and behaviors, particularly those characterized by creativity and innovation (Fredrickson and Branigan, 2005).Aligned with the Broaden-and-Build Theory, the second objective of this study is to elucidate the protective function of positive emotion (hope) as a potential mediator in the relationship between inner strength and self-management behavior among post-PCI patients.The following hypotheses were formulated: Hypothesis 1: Inner strength is positively related to self-management.Hypothesis 2: Inner strength is positively related to hope.Hypothesis 3: Hope is positively related to self-management.Hypothesis 4: Hope plays a mediating role between inner strength and self-management.The proposed model is constructed in Figure 1.The finding will enrich relevant theory and provide a new perspective for self-management in patients after PCI.
Study design and procedure
A cross-sectional study was conducted at Nanjing First Hospital from September 2022 to February 2023.Paper questionnaires were distributed by two trained investigators with master's degrees when the patients were stable.The survey was conducted in the teaching classroom or patient ward of the Cardiology Department at the Nanjing First Hospital.At the start of the survey, the investigators explained the study's purpose and significance to the participants using unified terminology and informed the patients about the points to consider when completing the questionnaires.With their consent, patients were guided to complete the questionnaire anonymously.Each questionnaire took about 10-15 min to complete and was collected on the spot.Any confusion or mistakes made during the completion of the questionnaire were immediately clarified and corrected.According to the principle that the sample size for multiple linear regression should be at least 5-10 times the number of independent variables, and considering there were about 22 variables in this study, the estimated sample size was set at 220 subjects to account for potential non-respondents (Chow et al., 2018).
Participants
Convenience sampling was employed to recruit participants.Patients who underwent PCI at Nanjing First Hospital were recruited for the study.The inclusion criteria were: (a) at least 18 years old, (b) met the criteria of the Chinese Guidelines for Percutaneous Coronary Interventions (2016) and received PCI for the first time (Han, 2016), (c) able to provide written informed consent, (d) able to complete the questionnaire independently or with the assistance of the investigator.The exclusion criteria were: (a) patients diagnosed with psychiatric diseases, such as schizophrenia, (b) patients with other severe somatic diseases, such as multiple organ failure or malignant tumors, (c) patients unable to take care of themselves in daily life.A total of 220 questionnaires were distributed, and after eliminating the unqualified ones, 216 questionnaires were deemed valid.
The self-designed general information questionnaire
The collected information included age, gender, marital status, education level, monthly family income, course of coronary heart disease and number of complications.
Inner strength scale
The Inner Strength Scale (ISS), developed by Lundman et al. (2011) based on their metatheoretical analysis, was used to assess the capacity that supports positive movement through lifechallenging events.It is a 20-item scale with four dimensions: firmness, creativity, connectivity, and flexibility.The ISS was Frontiers in Psychology 03 frontiersin.orgTheoretical model and hypotheses.
validated in Chinese among patients living with chronic illness (Hu et al., 2022).Courage was added as a new factor to the original scale, and the revised ISS contained five dimensions with a total of 20 items.Participants rated each item on a 6-point Likert scale (from 1 "completely disagree, " to 6 "completely agree").The total score of ISS, which ranged from 20 to 120, covered the sum of each item.Higher scores indicated stronger inner strength of patients.ISS has been widely used in the elderly population in China with satisfactory reliability and validity (Yu et al., 2018;Hu et al., 2022).The Cronbach's alpha coefficient of ISS in the present study was 0.914.
Herth hope index
The Herth Hope Index (HHI), compiled by Herth (1991), was applied to assess the positive attitudes toward uncertain events such as illness.The Chinese version was translated by Chan et al. (2012).HHI consists of 12 items in three dimensions: temporality and future (T), positive readiness and expectancy (P), and interconnectedness (I).Participants rated each item on a 4point Likert scale (from 1 "completely disagree, " to 4 "completely agree").The total score of HHI ranged from 12 to 48, and higher scores indicated higher levels of hope.The HHI is suitable for the assessment of hope in healthy populations, cancer, and other chronic diseases (Smith et al., 2019).In addition, the HHI has been demonstrated to be a reliable and valid instrument for evaluating hope in patients with heart disease (Chan et al., 2012).The Cronbach's alpha coefficient of ISS in the present study was 0.867.
Coronary self-management scale
The Coronary Self-Management Scale (CSMS) was compiled by Ren et al. (2009) and used to evaluate the management of related health behaviors in patients with coronary heart disease in China.The CSMS consists of seven dimensions, including daily life management, adverse habits management, disease knowledge management, symptom management, first aid technology management, treatment compliance management, and emotional cognition management, with a total of 27 items.Participants rated each item on a 5-point Likert scale (from 1 "never, " to 5 "always").The total score of CSMS ranged from 27 to 135, and higher scores indicated higher levels of selfmanagement.Currently, this scale is widely employed in China for the measurement of self-management behavior in CHD patients with good reliability and validity (Zhang et al., 2021;Zhu et al., 2022).The Cronbach's alpha coefficient of CSMS in the study was 0.943.
Data analysis
SPSS 26.0 was used for data processing and analysis.Prior to data analysis, the Shapiro-Wilk test was performed to check the normal distribution of the data.Continuous variables (ISS, HHI, CSMS scores) were normally distributed and described by mean ± standard deviation (SD).Categorical variables (age, gender, marital status, education level, monthly family income, course of coronary heart disease, and complications) were represented by frequency and percentage (%).The t-test or oneway analysis of variance was used to compare the differences in inner strength, hope, and self-management of CHD patients with different demographic characteristics.Pearson correlation analysis was performed to determine the correlation among inner strength, hope, and self-management in patients with CHD.A two-sided P < 0.05 was considered statistically significant.The mediating role of hope in the relationship between inner strength and self-management was tested using the bootstrap method (5,000 runs) by running the PROCESS macro 3.4 (model 4) developed by Hayes (2017) in SPSS.If 95% confidence intervals (CIs) did not include zero, then the effect was regarded as significant.
Common method bias
Common method bias was tested using Harman's single-factor test.The results showed that 11 factors with eigenvalues over 1 were obtained by unrotated principal component factor analysis.The variance explained by the first factor was 37.92%, less than the critical value of 40.00%, indicating that there was no serious common method bias in this study.
Comparison of inner strength, hope, and self-management scores in PCI patients with different demographic characteristics
The demographic characteristics of the research variables are shown in Table 1.Of the 216 respondents, 133 (61.6%) were males.The mean age ( ± SD) of the patients was 63.14 ± 10.75 years, ranging from 32 to 86 years.The majority of the participants were married (n = 207, 95.8%).About 33.8% of the patients had a middle school education.Half of the patients had a monthly family income ranging from 5,000 to 10,000 yuan (n = 108, 50.0%).Approximately 63.8% of the patients had a disease duration of less than 0.5 years.A total of 186 (86.1%) patients had at least one complication.As shown in Table 1, there were significant differences in the scores of hope and inner strength among patients with different genders, ages, education levels, and marital statuses (all P < 0.05).There were significant differences in the scores of self-management among patients with different ages, education levels, and disease durations (all P < 0.05).
Correlations between inner strength, hope, and self-management among patients after PCI
The mean scores of ISS, HHI, and CSMS were 81.46 ± 12.00, 35.94 ± 5.38, and 86.79 ± 14.84, respectively.As shown in Table 2, Pearson correlation analysis revealed that the total score of ISS was significantly and positively correlated with the total scores of HHI and CSMS (r = 0.867 and 0.630, respectively; all P < 0.05).Additionally, the total score of HHI was significantly and positively correlated with the total score of CSMS (r = 0.671, P < 0.05).
Analysis of the mediating effects of hope between inner strength and self-management
As shown in Table 3, the first step was to examine the effect of inner strength on self-management.The result showed that inner strength had a significant effect on self-management (β = 0.630, P < 0.01).The second step was to examine the effect of inner strength on hope.The result showed that inner strength had a significant on hope (β = 0.867, P < 0.01).The third step was to include inner strength and hope as independent variables in the regression equation to examine their impact on self-management.The result showed that the influence of inner strength on self-management was not significant after introducing hope as a mediating variable (β = 0.194, P = 0.057), suggesting that hope had a complete mediating effect between inner strength and self-management.To further determine whether the mediating effect coefficient was significant, the Bootstrap method was used to verify the confidence interval of the mediating effect.Five thousand samples were randomly sampled from the original data (n = 216), and the average of the path coefficient was calculated.As shown in the Table 4, the 95% confidence interval of the direct effect of inner strength on self-management contains 0 (−0.006∼0.394),while the 95% confidence interval of the mediating effect of hope does not contain 0 (0.285∼0.601), indicating that the mediation effect of hope is significant and completely mediating.The mediating effect (0.436) accounts for 69.21% of the total effect (0.630).The mediation effect model is shown in Figure 2.
Discussion
This study investigated the status of self-management, hope, and inner strength in patients after PCI and, to our knowledge, for the first time, tested the mediating role of hope between inner strength and self-management.Several analyses were carried out for the research question, including descriptive statistics, correlation, and mediation analysis.
Our findings revealed that self-management of patients after PCI was at a moderate level (86.79 ± 14.84), which was similar to the results obtained by Ma et al. (2018) in the study of self-management of patients after PCI in China.General life management had the highest item average score (3.38 ± 0.68), while emotional cognition management had the lowest item average score (2.98 ± 0.77), followed by treatment compliance management (3.02 ± 0.70) and bad habit management (3.17 ± 0.66).These results indicate that compared with understanding a healthy diet and appropriate activities in daily life, patients after PCI have more difficulties in long-term self-management behavior, changing bad habits such as smoking, and emotional regulation after an illness.This suggests that medical workers and family members should strengthen the supervision of the health behavior of such patients, promote the improvement of compliance management, and also pay attention to psychological counseling (Zhu et al., 2022).In addition, the difference analysis of self-management scores of patients with different socio-demographic characteristics showed that the higher the education level and the longer the course of the disease, the higher the level of self-management scores.This was consistent with previous studies (Lai et al., 2021).However, this study showed a negative association between age and self-management, different from the umbrella review results of Alexandre, which may be related to the relatively high literacy of younger patients in this study (Alexandre et al., 2021).On the one hand, compared with older patients, younger patients have better physical function and find it easier to complete the management of symptoms and general life.On the other hand, younger, more educated patients with a longer disease course may have a better ability to acquire, understand, and apply healthrelated information, more experience in coping with the impact of the disease, and therefore better self-management ability (Guo and Harris, 2016;Alexandre et al., 2021;Lai et al., 2021).
To our knowledge, there has been no quantitative study on inner strength in patients after PCI.In this study, the ISS of patients was (81.46 ± 12.00), which was at the medium level, similar to the chronic population (Hu et al., 2022).However, it was lower than Viglund's findings in the healthy elderly group (Viglund et al., 2013).Compared with healthy people, patients with CHD bear more burden of symptoms and economic pressure brought by the disease.Especially for those who undergo PCI for the first time, more attention is paid to treatment effect and prognosis in the short term, so it is easy to overlook the existence and application of individual internal resources.In this study, the HHI of patients was (35.94 ± 5.38), similar to the results of Zhang, and higher than that of the tumor population (Li et al., 2021;Zhang et al., 2021;Wnuk, 2022).As a Chinese saying goes, "Turn pale at the mention of cancer, " influenced by traditional concepts, patients have less fear of CHD, which is a common chronic disease in their mind, than cancer.In addition, most patients in this study had a disease course within 1 year after PCI (185 cases, 85.6%), so they have greater expectations for prognosis and control of disease progression.The present study found that patients' inner strength and hope scores after PCI varied by gender, age, education level and marital status.Compared with males, A mediation model depicting the impact of hope in the relationship between inner strength and self-management among patients after PCI.
females are more psychologically vulnerable and more likely to have negative emotions, so their internal resources are relatively weak (Shimamoto and Rappeneau, 2017).Moreover, high educational level and married status have a maintenance and promoting effect on inner strength and hope, which may be related to the fact that such patients have more scientific channels to acquire disease knowledge, better self-regulation ability, more social support and perception, and thus have more belief and hope to overcome the disease (Viglund et al., 2013).Correlation analysis showed that there was a positive correlation between the inner strength score and self-management score of patients after PCI (r = 0.630, P < 0.01).This result adds quantitative evidence to the view that the improvement of the self-management ability of chronic patients is the result of inner strength in the theoretical model of inner strength proposed by Dingley and Roux (2014).According to Dingley's theoretical model, patients with higher inner strength can face up to challenging life events and learn from the experience in the process of illness.By establishing connections with themselves, family and friends, the patients continuously optimize the use of internal and external resources, and adopt familiar behaviors (such as changing their diet, reasonable exercise, adherence to medication, etc.) to make positive self-adjustment to reach a new normal.The results of this study showed that the inner strength of patients after PCI was positively correlated with the level of hope, which was similar to the results of Borges et al. (2021).Patients with higher levels of inner strength are more inclined to view life after illness from a positive perspective, thus more likely to adjust their mentality to accept the disease and have more confidence and hope for physical and mental recovery (Zhang, 2021).The results of this study show that patients' hope level is positively associated with self-management, indicating that patients with high hope levels are more likely to adhere to treatment and effectively self-manage, which is consistent with previous findings (Snyder, 2002).As a positive internal dynamic force, hope is an important strategy for patients to cope with the disease.Existing studies show that a good level of hope can mobilize people's positive emotions and self-efficacy (Davidson et al., 2020).Patients with a higher level of hope are better able to resist the harm brought by negative emotions and have expectations and persistence for the treatment and rehabilitation of the disease.Therefore, they have a strong belief in the successful implementation and completion of a certain behavioral goal of self-management, which makes patients more willing to actively learn and cooperate with others to take positive actions to deal with the disease and improve their physical condition.Based on the above results, healthcare providers should value the positive significance of intrinsic strength and hope for patient behavior change.Nursing staff can encourage patients to face up to the disease through health education or psychological intervention, correct their misconceptions such as "illness = disability" and "PCI = cure, " and help patients realize the role of individual power in delaying the disease process, so as to improve the plasticity and enthusiasm of patients' self-management.
In this study, the analysis of mediation effects showed that the level of hope is a mediator variable of inner strength and self-management and acts as a complete mediator.This indicates that inner strength after PCI can influence and predict patients' self-management ability through the level of hope.Patients with higher inner strength have more core resources to resist disease and show stronger psychological adaptation and spiritual growth in the process of coping with the disease.Patients with inner strength do not deny the fact of illness, but regard the disease as normal, and have hope for the treatment and recovery of the disease in the process of actively accepting the disease.Driven by the positive spiritual force of hope, the patient's health responsibility is stimulated, and they are more inclined to take the initiative to deal with negative emotions, make use of social resources, and exercise self-control to achieve good self-management.Therefore, while providing objective support such as health knowledge and medical care for patients after PCI, medical staff should pay attention to the positive effect of patients' psychological capital on behavior.According to Snyder's hope theory, a nursing plan can be formulated from three aspects: goal, path thinking, and dynamic thinking (Ge et al., 2021).For example, when developing self-care strategies for patients, the whole process should be phased, and the patients can jointly agree on phased goals, starting from simple short-term goals, so that patients can have a positive experience of "I can" in the completion process.They can adjust their cognition of disease events, and treat disease as an opportunity to pay attention to their own health and make positive changes, encourage patients to pay attention to their own value in health promotion, and help patients to explore and use their own advantages to actively participate in the self-management of disease.
Limitations of the study
While the results of this study strongly supported the hypothesis of the mediating role of hope between inner strength and self-management, several limitations of the study must be acknowledged.First, this study adopts a cross-sectional design, so the causal relationship between variables should be considered carefully.Future research could conduct longitudinal studies to further verify the results of this model.Second, all the samples in this study were from a third-class A hospital in Nanjing, Jiangsu Province, which has the leading percutaneous coronary intervention technology in China.Patients who sought treatment here may have relatively high levels of hope and self-management awareness, leading to a certain degree of sampling bias.Therefore, the results of this study may not be extended to other coronary heart disease populations.Future research needs to be conducted on a larger scale and in multiple locations.Third, the disease course of most patients included in this study is within half a year, and the survival time of patients with the disease is not long.With the passage of time, patients' views on disease and disease management may change.Therefore, it is necessary to conduct longitudinal studies to explore the dynamic changes in patients' psychological experience and self-management.Moreover, this study only analyzed the relationship between inner strength, hope, and self-management through a simple mediation model.The exploration of mechanisms is far from sufficient, and in the future, more scientific approaches such as mediation chains, moderation effects, etc., should be employed for a more comprehensive and in-depth investigation into the mechanisms through which inner strength influences self-management.
Conclusion
This study demonstrated that the self-management, inner strength, and hope of patients after PCI were all at a moderate level, and there was still room for further improvement.The concepts were pairwise correlated, and hope played a complete mediating role between inner strength and self-management.First of all, self-management health education for PCI patients still needs to be strengthened, especially for the elderly, the patients with low education and a short course of disease.In addition, there are currently only a few interventional studies on patients' inner strength.In the future, while exploring intervention strategies related to inner strength, medical staff may take improving patients' hope level as the perspective and intervention target, so as to help patients rekindle their hope for life, accumulate internal resources, and actively participate in self-management.
TABLE 1
Comparison of ISS, HHI, and CSMS with different demographic characteristics.
10.3389/fpsyg.2024.1268598TABLE3Analysis of mediating effect of hope between inner strength and self-management. | 2024-01-26T16:31:54.529Z | 2024-01-24T00:00:00.000 | {
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233792976 | pes2o/s2orc | v3-fos-license | Pervaporation of Aqueous Ethanol Solutions through Rigid Composite Polyvinyl-Alcohol/Bacterial Cellulose Membranes
: The paper focuses on synthesis, characterization and testing in ethanol-water separation by pervaporation of new membrane types based on polyvinyl alcohol (PVA) and bacterial cellulose (BC). A technology for obtaining these membranes deposited on a ceramic support is presented in the experimental section. Three PVA-BC composite membranes with different BC content were obtained and characterized by FTIR, SEM and optic microscopy. The effects of operating temperature (40–60 ◦ C), permeate pressure (18.7–37.3 kPa) and feed ethanol concentration (24–72%wt) on total permeate flow rate (0.09–0.23 kg/m 2 /h) and water/ethanol selectivity (5–23) were studied based on an appropriate experimental plan for each PVA-BC membrane. Statistical models linking the process factors to pervaporation performances were obtained by processing the experimental data. Ethanol concentration of the processed mixture had the highest influence on permeate flow rate, an increase in ethanol concentration leading to a decrease in the permeate flow rate. All 3 process factors and their interactions had positive effects on membrane selectivity. Polynomial regression models were used to assess the effect of BC content in the dried membrane on pervaporation performances. Values of process performances obtained in this study indicate that these membranes could be effective for ethanol-water separation by pervaporation.
Introduction
The most attractive point of the pervaporation process is the separation of the organic solvents [1][2][3], especially the water removal. It presents a high level of performance and it is economically efficient. In the field of solvents dehydration, there have been many cases in which pervaporation technology has been employed and successfully implemented on an industrial scale [4,5]. Currently, more than 90 industrial units are in operation world-wide for the dehydration of ethanol, isopropanol, ethyl acetate and multipurpose solvents [6].
There are many studies focused on the development of new membranes that can be used for the pervaporation process as well as for other processes involving membranes. For the pervaporation process, the focus is on the enhancement of membranes, in order to be more efficient, with longer-term stability and life, and on the economic factor, expressed in terms of the accepted cost. Although many types of materials have been used in the development of membranes, the composite membranes based on polyvinyl-alcohol (PVA) are the ones mostly used in the pervaporation process [7][8][9][10][11]. Due to its numerous hydroxyl groups (Figure 1), PVA exhibits a high water permeability. Moreover, dried PVA-based membranes have better abrasion resistance, mechanical properties and selectivity for hydrophilic species [12]. The high degree of swelling of PVA determines an increase in the free volume of its network and a decrease in the chemical interactions with the permeating species, resulting in low membrane selectivity and stability [12]. Various techniques have been employed to obtain enhanced performances of PVA membranes [13][14][15][16]. Praptowidodo [13] studied the influence of the cross-linking degree on PVA-based membrane performances by adding glutaraldehyde (GA) as a cross-linking agent. An increase in the amount of GA led to a decrease in the degree of swelling, resulting in a decrease in the permeate flow rate and an improvement of membrane selectivity. The mechanical properties of composite membranes based on PVA can be highly improved by adding suitable reinforcement materials in PVA. Huang et al. [17] prepared a composite hydrogel containing PVA and polyvinyl pyrrolidone (PVP) with wrapped multiwalled carbon nanotubes. Unlike a PVA-based membrane, this composite membrane exhibited about a 133% increase in the tensile strength and 63% in the tear strength.
Bacterial cellulose (BC) is a special cellulose with a nanoscale structure, produced by selected aerobic Gram-negative bacteria, which are active on saccharide substrates at a certain composition, pH and temperature [18]. BC is a very pure product, with a high degree of crystallinity and polymerization and with an interesting mechanical behavior [19,20]. BC exhibits superior structural properties compared to plant cellulose [21]. It has been proven that BC, either pure or composite, can be used as a valuable biopolymer in various fields (e.g., industrial, medical, food processing) [22][23][24]. The hydrophilic nature of pure BC and BC-based composites confers these types of membranes selectivity in water transport, if a binary mixture containing water and another compound passes through the membrane. During the BC membrane pervaporation process of binary ethanol-water mixture, as the water content is high, it results in a high permeate flow rate and a low selectivity.
Recently, much research has been focused on PVA-BC composites, especially because both components present strong hydrophilic behavior. In PVA-BC composites, BC appears as fiber, film or bulk material. The aim is to obtain a membrane with good mechanical properties, high chemical stability as well as good selectivity and permeability for transferable species [13]. For PVA-BC nanocomposite layers, Gea et al. [25] presented different preparation methods; the results showed that the presence of PVA led to a reduction in Young's modulus and in an increase in toughness compared to a pure BC layer. Tan et al. [26] and Chiciudean et al. [27,28] used a layered PVA-BC composite hydrogel, which exhibited excellent mechanical properties.
High permeate flow rates and acceptable water selectivities were obtained for pervaporation of ethanol-water mixtures through BC membranes [29]. However, due to membrane cracking at drying and also excessive swelling, a high pervaporation surface is not possible. Pervaporation performances of BC-PVA membranes, obtained by impregnating BC gel with PVA and cross-linking with GA [30], were superior to those of BC membranes. However, these types of membranes have the same disadvantage of cracking and swelling, which does not allow the development of pervaporation surfaces required by applications with higher working capacity. Various techniques have been employed to obtain enhanced performances of PVA membranes [13][14][15][16]. Praptowidodo [13] studied the influence of the cross-linking degree on PVA-based membrane performances by adding glutaraldehyde (GA) as a cross-linking agent. An increase in the amount of GA led to a decrease in the degree of swelling, resulting in a decrease in the permeate flow rate and an improvement of membrane selectivity. The mechanical properties of composite membranes based on PVA can be highly improved by adding suitable reinforcement materials in PVA. Huang et al. [17] prepared a composite hydrogel containing PVA and polyvinyl pyrrolidone (PVP) with wrapped multiwalled carbon nanotubes. Unlike a PVA-based membrane, this composite membrane exhibited about a 133% increase in the tensile strength and 63% in the tear strength.
Bacterial cellulose (BC) is a special cellulose with a nanoscale structure, produced by selected aerobic Gram-negative bacteria, which are active on saccharide substrates at a certain composition, pH and temperature [18]. BC is a very pure product, with a high degree of crystallinity and polymerization and with an interesting mechanical behavior [19,20]. BC exhibits superior structural properties compared to plant cellulose [21]. It has been proven that BC, either pure or composite, can be used as a valuable biopolymer in various fields (e.g., industrial, medical, food processing) [22][23][24]. The hydrophilic nature of pure BC and BC-based composites confers these types of membranes selectivity in water transport, if a binary mixture containing water and another compound passes through the membrane. During the BC membrane pervaporation process of binary ethanol-water mixture, as the water content is high, it results in a high permeate flow rate and a low selectivity.
Recently, much research has been focused on PVA-BC composites, especially because both components present strong hydrophilic behavior. In PVA-BC composites, BC appears as fiber, film or bulk material. The aim is to obtain a membrane with good mechanical properties, high chemical stability as well as good selectivity and permeability for transferable species [13]. For PVA-BC nanocomposite layers, Gea et al. [25] presented different preparation methods; the results showed that the presence of PVA led to a reduction in Young's modulus and in an increase in toughness compared to a pure BC layer. Tan et al. [26] and Chiciudean et al. [27,28] used a layered PVA-BC composite hydrogel, which exhibited excellent mechanical properties.
High permeate flow rates and acceptable water selectivities were obtained for pervaporation of ethanol-water mixtures through BC membranes [29]. However, due to membrane cracking at drying and also excessive swelling, a high pervaporation surface is not possible. Pervaporation performances of BC-PVA membranes, obtained by impregnating BC gel with PVA and cross-linking with GA [30], were superior to those of BC membranes. However, these types of membranes have the same disadvantage of cracking and swelling, which does not allow the development of pervaporation surfaces required by applications with higher working capacity.
In this paper, we assess PVA-BC membranes, with BC distributed discreetly and controlled in PVA, applied on supports of any shape and size, and processed by crosslinking with total water removal, cover all practical requirements of a pervaporation process with water transport. Accordingly, hydrophilic fibers of BC were mixed with PVA and GA and the mixture was deposited on a ceramic support, to obtain rigid PVA-BC membranes with a high operational effectiveness when used for water separation from various mixtures. The main aim of the paper was the study of pervaporation performances during the dehydration of ethanol-water solutions using rigid PVA-BC composites deposited on a ceramic support.
Materials
The pervaporation rigid membranes were prepared from PVA powder type M w 89,000-98,000 (Sigma Aldrich), BC gel (0.995 g/cm 3 ) having 96% water, fibrils with diameters less than 50 nm, a crystallinity index of 78−90% and a polymerization degree of 2500−4000 (UPB Mass Transfer Laboratory) and GA (Merck) as a cross-linking agent. The membranes were deposited on a high porosity ceramic tube support (type TC-00314, Research, Design and Production Center for Refractive (CCPPR), Alba Iulia, Romania).
Membrane Preparation
There were two steps involved in preparing the disperse systems used to obtain PVA-BC membranes. The first step involved the preparation of a PVA solution having a PVA content of 75 g/L (by dissolving 12 g of PVA in 150 mL of distilled water, under moderate agitation, at 60 • C. In the second step, BC gel was ground by using a disintegrator knife and then mixed with the PVA solution to obtain a PVA/BC ratio of 2 (12 g PVA and 6 g BC), 1 (12 g PVA and 12 g BC) and 0.66 (12 g PVA and 18 g BC), respectively. Thus, three PVA-BC disperse systems were obtained, the concentrations of PVA and BC being 7.14% and 3.57% for the first system (S1), 6.90% and 6.90% for the second system (S2), 6.67% and 10% for the third system (S3), respectively. The disperse systems were stored at 5 • C before being used.
Rigid PVA-BC membranes were obtained as follows: (i) the membrane support (ceramic tube) was cleaned with distilled water, dried at 105 • C and then weighed; (ii) 30 g of disperse system (S1, S2 or S3) was mixed with 1.2 g of GA, which was used as a cross-linking agent; (iii) the membrane mixture was applied on the ceramic tube with a brush; after the first layer was applied, the system was dried at 60 • C for 15-20 min and then weighed; the second layer was applied and the procedure was repeated until the thin layer reached the desired mass (1.3-1.8 g on the ceramic support with an external diameter of 0.035 m and a length of 0.25 m); (iv) supported membrane was dried at 90 • C for 90-120 min, then weighed to determine the final mass and thickness of the active membrane layer. Depending on the disperse system (S1-S3) from which they were prepared, the membranes were coded as follows: PVA-BC 12/6 (M1), PVA-BC 12/12 (M2) and PVA-BC 12/18 (M3). Figure 2 shows the stages of preparation of rigid PVA-BC membranes applied on the ceramic tube. The computed thickness of membranes was about 45 µm, according to some tests performed with a PosiTector 6000 apparatus (DeFelsko inspection Instruments). In order to have membrane samples for physicochemical analysis (FTIR, SEM), film samples were applied on a glass plate.
Pervaporation Tests
Pervaporation experiments involving these rigid membranes were carried out in a laboratory equipment (Figure 3). The membrane (1b) deposited on the ceramic support (1c) and the glass tube (1a) of the pervaporation unit (1) were heated by an electric resistance heater, the heating eliminating the permeate condensation on the glass tube. The pervaporation chamber was connected to the vacuum and to the systems for measur-
Pervaporation Tests
Pervaporation experiments involving these rigid membranes were carried out in a laboratory equipment ( Figure 3). The membrane (1b) deposited on the ceramic support (1c) and the glass tube (1a) of the pervaporation unit (1) were heated by an electric resistance heater, the heating eliminating the permeate condensation on the glass tube. The pervaporation chamber was connected to the vacuum and to the systems for measuring/control the process temperature and pressure as well as the permeate flow rate.
Pervaporation Tests
Pervaporation experiments involving these rigid membranes were carried out in a laboratory equipment ( Figure 3). The membrane (1b) deposited on the ceramic support (1c) and the glass tube (1a) of the pervaporation unit (1) were heated by an electric resistance heater, the heating eliminating the permeate condensation on the glass tube. The pervaporation chamber was connected to the vacuum and to the systems for measuring/control the process temperature and pressure as well as the permeate flow rate. As shown in Figure 3, the ethanol-water mixture (1e) was placed in the precision burette (6), which was connected to the liquid pervaporation chamber (1). The liquid mixture inside the pervaporation unit passed through the membrane, where the water was preponderantly removed and the ethanol was concentrated in the pervaporation system. On the external side of the membrane, the water and the associated ethanol were vaporized due to the low pressure on the membrane surface. The vapor phase was condensed in a cooling system, which consists of two parts: the first part is a glass coil refrigerator working at a temperature of 5-6 °C, whereas the second part is a cold trap containing ice. The purpose of using this cooling system is to provide as much cold space as possible for the condensation of all permeating vapor.
Pervaporation tests were performed at different levels of temperature (40, 50 and 60 °C), pressure (140, 210 and 280 mm Hg, i.e., 18.7, 28.0 and 37.3 kPa) and ethanol concentrations in the feed solution (24, 48 and 72%wt). The ethanol concentrations in the feed and in the condensed permeate were determined by measuring the refractive index. As shown in Figure 3, the ethanol-water mixture (1e) was placed in the precision burette (6), which was connected to the liquid pervaporation chamber (1). The liquid mixture inside the pervaporation unit passed through the membrane, where the water was preponderantly removed and the ethanol was concentrated in the pervaporation system. On the external side of the membrane, the water and the associated ethanol were vaporized due to the low pressure on the membrane surface. The vapor phase was condensed in a cooling system, which consists of two parts: the first part is a glass coil refrigerator working at a temperature of 5-6 • C, whereas the second part is a cold trap containing ice. The purpose of using this cooling system is to provide as much cold space as possible for the condensation of all permeating vapor.
Pervaporation tests were performed at different levels of temperature (40, 50 and 60 • C), pressure (140, 210 and 280 mm Hg, i.e., 18.7, 28.0 and 37.3 kPa) and ethanol concentrations in the feed solution (24, 48 and 72%wt). The ethanol concentrations in the feed and in the condensed permeate were determined by measuring the refractive index.
Characteristic working procedure of a pervaporation test was as follows: (i) the ceramic tube of pervaporation unit was filled with ethanol-water mixture and the working liquid level in the burette was measured; (ii) working temperature and pressure levels were selected and the vacuum pump was started; (iii) the time (∆τ) after which the liquid level in the burette decreased by V = 2 mL was measured; (iv) the burette was refilled and other 3 measurements of ∆τ were performed; (v) the refractive index of the condensate collected in the steam condenser was measured.
Process Parameters
Total permeate flow rate, N A , was determined using Equation (1), where A is the effective membrane area, ρ f the density of feed solution and ∆τ the time after which the liquid level in the burette decreased by V = 2 mL.
Ethanol concentrations in the feed and permeate samples were estimated using an Atago Abbe refractometer (Atago, Japan). Based on the experimental data obtained for the refractive index at 25 • C and refractive index-concentration curves [31], the mass fractions of ethanol were obtained. Water/ethanol selectivity, α w/et , was calculated using Equation (2), where X and Y represent the mass fractions of species in the feed and permeate.
Results and Discussions
Pervaporation data corresponding to 4 experimental runs (measurements of ∆τ) using PVA-BC 12/6 membrane (M1) are summarized in Table 1, where c et is the concentration of ethanol in the feed solution, t the temperature, p the pressure, V the volume of feed solution passed through membrane in the time ∆τ, n f and n p are the refractive indexes for the feed solution and permeate. Characteristic FTIR spectra of PVA-BC 12/6, PVA-BC 12/12 and PVA-BC 12/18 membranes used in the pervaporation experiments are shown in Figure 4. Due to the fact that the FTIR spectra only analyze the vibration of the molecular groups, it is not expected to have many differences among the spectra of membranes, which differ only in BC content. For a deeper analysis, the FTIR spectra for BC, PVA and PVA cross-linked with GA are provided ( Figures 5 and 6).
According to Figures 5 and 6 (C-O stretching, alcohol). Also, a peak at 1629 cm −1 (C-OH absorbed water), can be identified in the FTIR spectrum of BC ( Figure 5) and similar peaks, but of much lower intensity, appear in characteristic FTIR spectra of PVA-BC membranes (Figure 4), i.e., at 1640.0 cm −1 for PVA-BC 12/6 and 1658.7 cm −1 for PVA-BC 12/18. The low intensity of these peaks, probably due to the heat treatment (60−90 min at 90 • C) to complete cross-linking, may indicate that these membranes do not trend to retain water and swell.
The spectra of composite membranes (M1-M3) shown in Figure 4 indicate a slight displacement of characteristic peaks of BC and PVA cross-linked with GA as well as the appearance of new peaks, specific to these membranes. The results plotted in Figure 4 highlight 7 peaks for PVA-BC 12/6 membrane (M1), 9 peaks for PVA-BC 12/12 membrane (M2) and 11 peaks for PVA-BC 12/18 membrane (M3). Accordingly, new peaks appear as the concentration of BC in the membranes increases. This suggests that each PVA-BC membrane has its own structure, which depends on BC content. It is expected that the Processes 2021, 9, 437 6 of 14 behavior of the membranes in the pervaporation process will depend on the BC content in the membrane, which will be considered as a process factor in the future.
trend to retain water and swell.
The spectra of composite membranes (M1-M3) shown in Figure 4 indicate a slight displacement of characteristic peaks of BC and PVA cross-linked with GA as well as the appearance of new peaks, specific to these membranes. The results plotted in Figure 4 highlight 7 peaks for PVA-BC 12/6 membrane (M1), 9 peaks for PVA-BC 12/12 membrane (M2) and 11 peaks for PVA-BC 12/18 membrane (M3). Accordingly, new peaks appear as the concentration of BC in the membranes increases. This suggests that each PVA-BC membrane has its own structure, which depends on BC content. It is expected that the behavior of the membranes in the pervaporation process will depend on the BC content in the membrane, which will be considered as a process factor in the future. The spectra of composite membranes (M1-M3) shown in Figure 4 indicate a slight displacement of characteristic peaks of BC and PVA cross-linked with GA as well as the appearance of new peaks, specific to these membranes. The results plotted in Figure 4 highlight 7 peaks for PVA-BC 12/6 membrane (M1), 9 peaks for PVA-BC 12/12 membrane (M2) and 11 peaks for PVA-BC 12/18 membrane (M3). Accordingly, new peaks appear as the concentration of BC in the membranes increases. This suggests that each PVA-BC membrane has its own structure, which depends on BC content. It is expected that the behavior of the membranes in the pervaporation process will depend on the BC content in the membrane, which will be considered as a process factor in the future. Optic microscope images of BC fibrils in PVA solutions are shown in Figure 7. We observed the BC distribution as micro agglomerates with concentrations consistent with BC content in the mixture. SEM images presented in Figure 8 show composites membrane in which the com- Optic microscope images of BC fibrils in PVA solutions are shown in Figure 7. We observed the BC distribution as micro agglomerates with concentrations consistent with BC content in the mixture. SEM images presented in Figure 8 show composites membrane in which the compact PVA film is furrowed by BC fibers having diameters less than 2 µm. It appears that the density of fibrils in the films is proportional to the BC content of the disperse system used to prepare the membranes. Optic microscope images of BC fibrils in PVA solutions are shown in Figure 7. We observed the BC distribution as micro agglomerates with concentrations consistent with BC content in the mixture. SEM images presented in Figure 8 show composites membrane in which the compact PVA film is furrowed by BC fibers having diameters less than 2 μm. It appears that the density of fibrils in the films is proportional to the BC content of the disperse system used to prepare the membranes. Pervaporation tests for each membrane used in the experimental study were organized according to a factorial plan with 2 levels for each factor, i.e., pervaporation temperature (t), pervaporation pressure (p) and ethanol concentration in the feed solution (cet). Process response variables in terms of permeate flow rate and membrane selectivity Pervaporation tests for each membrane used in the experimental study were organized according to a factorial plan with 2 levels for each factor, i.e., pervaporation temperature (t), pervaporation pressure (p) and ethanol concentration in the feed solution (c et ). Process response variables in terms of permeate flow rate and membrane selectivity depending on process factors for PVA-BC 12/12 membrane (M2) are summarized in Table 2. The dimensionless factors (x 1 , x 2 and x 3 ) were calculated using Equations (3)-(5), where the c subscript indicates the center of experimental plan. Characteristic experimental data matrices of 2 3 factorial experiments for PVA-BC 12/6 (M1), PVA-BC 12/12 (M2) and PVA-BC 12/18 (M3) membranes, containing the values of process responses at various levels of dimensionless factors, are given in Tables S1-S3. Levels of process response variables for all pervaporation measurements, i.e., 0.09-0.23 kg/m 2 /h for total permeate flow rate and 5-23 for water/ethanol selectivity, are summarized in Figure 9. These levels are in the same ranges as those characterizing the cellulose-silica membranes [21,32] or some special cellulose membranes [33,34].
( ) Data presented in Figure 9 were used to quantify the effect of pervaporation factors on response variables. Experimental data for each membrane (M k , k = 1..3) were processed as follows: (i) characteristic regression analysis of a 2 3 experimental plan was used to obtain values of significant regression coefficients in dependences N Ak (x 1 ,x 2 ,x 3 ) and α w/etk (x 1 ,x 2 ,x 3 ) given by Equations (6) and (7) [35]; (ii) significant regression coefficients in Equations (6) and (7), β ik and α jk , were processed resulting in 2 polynomial regression models, β i (x 4 ) = b 0i + b 1i x 4 + b 2i x 4 2 and α j (x 4 ) = a 0j + a 1j x 4 + a 2j x 4 2 , where x 4 represents BC concentration in the dried membrane; analytical expressions N A (x 1 ,x 2 ,x 3, x 4 ) and α w/et (x 1 ,x 2 ,x 3, x 4 ) were determined based on these polynomial regression models.
Equations (8)-(13) highlight the following issues: (i) N A1 , N A2 and N A3 , which are expressed by Equations (8), (10) and (12), depend linearly on temperature (x 1 ) and ethanol concentration in the feed mixture (x 3 ) and x 3 has a higher effect; an increase in x 1 and a decrease in x 3 lead to an increase in N Ak (k = 1..3); for PVA-BC 12/12 membrane (M2), there is a low linear influence of the pressure from the pervaporation unit [the term + 0.0057x 2 in Equation (10)]; for PVA-BC 12/18 membrane (M3), the interaction between temperature and composition of processed mixture diminishes the permeate flow rate [the term − 0.0062x 1 x 3 in Equation (12)]; (ii) α w/et1 , α w/et2 and α w/et3 , which are expressed by Equations (9), (11) and (13), depend on process factors and their interactions; all regression coefficients are positive, consequently, an increase in x 1 , x 2 and x 3 leads to an increase in α w/etk (k = 1..3).
These findings are similar to those obtained for pervaporation of ethanol-water system or isopropanol-water mixture through BC [29], BC-PVA [30] or cellulose based membranes [38].
In order to validate the statistical models described by Equations (15) and (16), 5 experimental runs were performed at x 1 = x 2 = x 3 = x 4 = 0.5, i.e., t = 55 • C, p = 32.7 kPa, c et = 60%wt and c BC = 6%wt (PVA-BC 12/15 membrane). Experimental values of process performances for these replicates (n = 5), N A,ex and α w/et,ex , and those predicted by Equations (15) and (16), N A,pr and α w/et,pr , are summarized in Table 5. A test statistic for one sample T-test, denoted T and defined by Equation (17), was calculated for each process performances (P) to assess whether the difference between the mean of experimental values and predicted value (P ex.mn − P pr ) is statistically significant. Values of P ex.mn , standard deviation (SD), standard error of the mean (SE), coefficient of variance (CV = 100SD/P ex.mn ), degrees of freedom (df = n − 1) and T are specified in Table 5. The difference P ex.mn − P pr is statistically non-significant for each process performance [the values of T (1.147 and 1.236) are lower than its critical value for two tails at a significance level of 0.05 and df = 4, i.e., T cr (0.05,4) = 2.776], which demonstrates the model validity. Accordingly, both Equations (15) and (16) can be used to design and simulate pervaporation units equipped with supported PVA-BC membranes, at levels of process factors in the ranges selected in the experimental study. T = P ex,mn − P pr SD √ n = P ex,mn − P pr SE (17) Table 5. Experimental and calculated data for replicates used to validate the statistical models described by Equations (15) and (16).
Conclusions
The composite PVA-BC membranes deposited on ceramic supports can be very effective to remove water from solvents. Preparation, characterization and testing of rigid PVA-BC membranes applied on a ceramic support are presented in this paper. Three membranes types, i.e., M1, M2 and M3, with different concentration of BC in the dried membrane (2.4, 4.8 and 7.2%wt) were prepared and then tested in pervaporation experiments. An experimental equipment at a laboratory pilot scale was used to study the pervaporation of ethanol-water mixtures through supported PVA-BC membranes.
A two-level experimental plan based on 3 factors, i.e., temperature (x 1 ), permeation pressure (x 2 ) and ethanol concentration in the feed solution (x 3 ), was used in the experimental investigation of the pervaporation behavior of each membrane type. Regression equations linking these 3 factors to process performances in terms of permeate flow rate (N Ak ) and membrane selectivity (α w/etk ) were obtained according to characteristic procedure of a 2 3 factorial experiment for each membrane type (M k , k = 1..3). Regression coefficients in these equations, β ik (i = 0, 1, 2, 3, 13) and α jk (j = 0, 1, 2, 3, 12, 23, 123), were processed resulting in 2 polynomial regression models, β i (x 4 ) = b 0i + b 1i x 4 + b 2i x 4 2 and α j (x 4 ) = a 0j + a 1j x 4 + a 2j x 4 2 , where x 4 represents BC concentration in the dried membrane. Analytical expressions N A (x 1 ,x 2 ,x 3, x 4 ) and α w/et (x 1 ,x 2 ,x 3, x 4 ) were determined based on these polynomial regression models. These relationships can be used to predict the pervaporation performances at levels of process factors in the ranges selected in the experimental study. | 2021-05-07T00:04:39.522Z | 2021-02-28T00:00:00.000 | {
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247245825 | pes2o/s2orc | v3-fos-license | Heat Stress Decreases Intestinal Physiological Function and Facilitates the Proliferation of Harmful Intestinal Microbiota in Sturgeons
Heat is a common source of stress in aquatic environments and can alter the physiological and metabolic functions of aquatic animals, especially their intestinal function. Here, the effects of heat stress on the structure and function of the intestine and the characteristics of the intestinal microbiota were studied in sturgeon (Acipenser baerii ♀ × Acipenser schrenckii ♂ hybrid F1). Sturgeons were exposed to sub-extreme (24°C) and extreme (28°C) high water temperatures for 12 days. The heat stress caused systemic damage to the intestine of sturgeons, which displayed severe enteritis in the valve intestine. The microbial diversity analysis showed that heat stress led to the disorder in intestinal microbiota, manifesting as an explosive increase in the abundance of thermophilic intestinal pathogens such as Plesiomonas, Cetobacterium, and Aeromonas and causing physiological dysfunction in the sturgeons. The disorder was followed by significant inhibition of intestinal digestion with reduced chymotrypsin, α-amylase, and lipase activities in the valve intestine and of antioxidant function with reduced peroxidase (POD) and catalase (CAT) activities. Simultaneously, heat stress reduced the thermal tolerance of sturgeons by reducing Grp75 expression and damaged the valve intestine’s repair ability with increased Tgf-β expression. The results confirmed that heat stress damaged the sturgeon intestines obviously and disturbed the intestinal microbiota, resulting in serious physiological dysfunction. The present study investigated the mechanism of the effect of heat stress on the sturgeon intestine and will help develop strategies to improve the resistance to thermal stress for wild and cultured sturgeons.
INTRODUCTION
Heat stress in local waters is likely to worsen due to global warming, threatening aquatic animals and potentially altering their behavior, growth, development, reproduction, and digestion (Hui-Huang et al., 2012;Miller et al., 2015;Aidos et al., 2020). Heat stress has severe consequences including death to fish (Yang et al., 2021). Heat stress threatens the survival of cold-water fishes, especially for sturgeons whose wellbeing closely relates to the environmental temperature. found that the water temperature in the Yangtze River basin, which is the primary habitat of Chinese sturgeons, has increased by 1 to 3.5 • C in autumn and 2 to 5 • C in winter over the past 60 years . Similarly, Larnier et al. (2010) found that the average surface water temperature in summer in Garonne, where the European sturgeon (Acipenser sturio) mainly lives, rose by 2.9 • C between 1978 and 2005 (Larnier et al., 2010). Temperature increases not only directly delay the spawning of sturgeons but also can lead to degeneration in gonad development . Affected sturgeons have been observed to have problems related to feeding (Mizanur et al., 2014), growth (Aidos et al., 2020), and reproduction, and in some cases, increased mortality has occurred (Lankford et al., 2003;Ficke et al., 2007;Hassell et al., 2008). The hybrid sturgeon (Acipenser baerii ♀ × Acipenser schrenckii ♂ hybrid F1) is a kind of commercial sturgeon valued globally for its flesh (Bronzi et al., 2019). Sturgeon habitats in China have historically been concentrated in the Yangtze and Yellow Rivers, but the temperatures of these rivers are rising (Liu et al., 2009;; Figure 1A). The abnormally high water temperatures from July to September present an especially difficult challenge for this cold-water fish; and numerous studies have associated immune decline, illness, and death with rising river temperatures (Shen et al., 2014;Castellano et al., 2017;. Therefore, the present study investigated the potential mechanisms by which heat stress influences the yields of commercial sturgeons. The intestine is the largest organ involved in the digestion and absorption of nutrients, as well as the largest organ involved in the immunity, defense, and endocrine systems in fish (Zhu et al., 2013). In freshwater fish, digestive enzyme activities in the intestinal mucosa of Carassius auratus, Cyprinus carpio, Rutilus, and Perca fluviatilis decreased under high water temperatures, and rapid increases in water temperature adversely affected the rate of carbohydrate hydrolysis and lowered the thermal tolerance of the intestinal tract (Golovanova et al., 2013). In addition, high water temperatures reduced the secretion of digestive enzymes in fish intestines, resulting in decreased intestinal chyme transport time and digestibility (Miegel et al., 2010;Tirsgaard et al., 2015). Stable gastrointestinal function contributes to the normal biological function of sturgeons and improves the yield and quality of the meat of commercial sturgeons, but the effects of heat stress on the structure and function of the sturgeon intestine are still unknown.
Intestinal microbiota in fish have dynamic compositions of aerobic bacteria, facultative anaerobes, and anaerobic bacteria (Evariste et al., 2019). The ecological balance of intestinal microbiota is important for maintaining normal feeding behavior, growth performance, digestive capability, and fecundity in fish (Kunz et al., 2009;Desai et al., 2016;Chenggang and Wenjing, 2017). Studies have shown that increased temperatures can disrupt the balance of intestinal microbiota and facilitate the growth of pathogenic bacteria. High water temperatures have been shown to trigger explosions of the pathogen Sphingomonas that can decimate sea cucumber populations (Zi-Jiao et al., 2019). Larios Soriano et al. (2018) confirmed that increased water temperatures resulted in increased abundance and richness of intestinal microbiota in the yellowtail kingfish Seriola lalandi, along with an increased abundance of a large number of harmful bacteria (Larios Soriano et al., 2018). A study of lake sturgeon (Acipenser fulvescens) found a dynamic relationship between the composition of intestinal microbiota and the physiology of the host gastrointestinal tract (Razak and Scribner, 2020). However, the effects of high water temperatures on the intestinal microbiota of cold-water fish like sturgeons remain to be studied.
This study investigated the composition of intestinal microbiota and physiological and biochemical indexes in the intestine of sturgeons under different water temperatures. It revealed the mechanism of the effect of heat stress on sturgeon intestines, and the results will provide guidance toward managing thermal stress in wild and cultured sturgeons.
Fish Maintenance and Treatment Protocols
All animal handling procedures were approved by the Animal Care and Use Committee of Sichuan Agricultural University in accordance with the Animal Experiment Guidelines of Sichuan Agricultural University, license no. ZCY-2019202031. For the experiment, a total of 180 6-month-old healthy juvenile sturgeons (A. baerii ♀ × A. schrenckii ♂ hybrid F1) of 76.37 ± 9.45 g and 23.54 ± 2.41 cm length were purchased from Sichuan Runzhao Fishery Co., Ltd., Sichuan, China.
Referring to the study of Mai et al. (2014), Bai et al. (2012), and the data collected at the farm, 18 to 20 • C was deemed to be the optimal temperature range for sturgeon growth (Bai et al., 2012;Mai et al., 2014). The fish were acclimated in the laboratory at 20 • C for 2 weeks prior to the experiment. The sturgeons were then randomly divided into three groups, a control group kept at 20 • C and two elevated temperature groups at 24 • C (G1) and 28 • C (G2) (Yang et al., 2021). Each group included four parallel tanks with 15 fish each. The experimental heating and heat treatment steps are illustrated in Figure 1B. The control group was held at 20 • C at room temperature for the duration of the experiment. The G1 and G2 groups were also held in room temperature water for the first 14 days, and then the temperature was increased by 1 • C/day until reaching the experimental temperatures of 24 and 28 • C on day 22 where the temperature was maintained until day 34. During the treatment period, the state of the experimental fish was continuously observed for 24 h. Oxygen was continuously supplied by the oxygen pumps for 24 h, and dead or dying fish were quickly removed.
During the experiment, the feeding rates and deaths of the sturgeons in each group were recorded daily, along with dissolved oxygen in the water. On day 34, upon completion of the heat treatment, the sturgeons in each group were euthanized with tricaine mesylate (MS-222) (Sigma-Aldrich, Beijing, China). Blood was collected via the caudal vein, and fish were dissected for biochemical examination. The histopathology, ultrastructure, enzyme activity, mRNA expression, and intestinal microbiota of gastrointestinal tissues and contents were examined. FIGURE 1 | Sturgeon production in part of the province in China in 2017 (Le-jun and Yong-hui, 2018), water temperature scheme applied to the sturgeon in the present study, effects of heat stress on water dissolved oxygen, sturgeon feeding rate, and cumulative survival rate. Sturgeon production in 2017 (A). During the experiment, each tank volume was 0.5 m 3 , dissolved oxygen was maintained above 7.5 mg/L, and pH was 7.4 ± 0.4. Feed was added twice a day (at a 7-h interval); the daily feed amount was 2% of the fish body mass. The water in the tanks was pretreated with UV light and an aeration process, and 25% of the culture water was renewed every day. Fish were held in a 12-h:12-h light/dark cycle
Examination of Anatomy, Histopathology, and Ultrastructure
The sturgeons from day 35 were euthanized and dissected for examination. The physical morphology of the gastrointestinal tract was observed. Tissue specimens of the intestines were fixed in 4% polyformaldehyde solution and, following a routine process, fixed in paraffin and stained with H&E. Histological slides were examined under a light microscope (Nikon, Tokyo, Japan). Intestinal tissue was placed in a fixative of 2.5% glutaraldehyde in pH 7.4 cacodylate buffer, washed three times in phosphate-buffered saline (PBS), and post-fixed with 1% osmium tetroxide. After dehydration in graded alcohol, the tissues were embedded in Araldite. The blocks were sectioned on a microtome with a glass knife. The 6.575-µm-thick sections were placed on uncoated copper grids, stained with uranyl acetate, and post-stained with 0.2% lead citrate. A transmission electron microscope (HT7700, Hitachi, Tokyo, Japan) was used to observe and collect images for analysis.
The degrees of hemorrhage, edema, deposits, hypertrophy, hyperplasia, atrophy, infiltration, and necrosis of the gastrointestinal tract were evaluated according to Huang et al. (2019). Every change was given an S score ranging from 0 to 6, depending on the degree and extent of the change: unchanged (0), mild change (2), moderate change (4), and severe change (6) as a diffuse lesion. Intermediate values were also considered. The organ index (I = t alt [S × ω IF ]) and total index (I = Org t alt [S × ω IF ]) were calculated for each experimental group in this study, where ω IF was the important factor.
Antioxidant Index and Digestive Enzyme Activity Determination
The antioxidant and digestive enzyme activity indexes for the intestine and serum were determined using diagnostic kits produced by Nan Jing Jian Cheng Bioengineering Institute (Nanjing, China) following the manufacturer's instructions. Approximately 0.1-g valve intestinal tissue was homogenized with 0.9 ml of 0.65% NaCl solution in a homogenizer on ice. The homogenates and whole blood were then centrifuged at 3,500 × g 10 min at 4 • C, and total protein in the supernatant of the tissue was determined with a protein quantification kit (A045-2). The diagnostic kits used in this experiment were for catalase (CAT) (A007-1), glutathione peroxidase (GSH-Px) (A005), peroxidase (POD) (A084-1), alpha amylase (α-AMS) (C016-1), LPS (A054-1), and chymotrypsin (CHY) (A080-3).
Real-Time Quantitative Polymerase Chain Reaction
Primer sequences for β-actin, ef1a, Hsp60, Hsp70, Tgf-β, and Grp75 were designed and synthesized based on unpublished transcriptome data by the Beijing Qingke Biotech Co., Ltd. (Beijing, China). The housekeeping genes β-actin and ef1a were used as internal references. All primer sequences are shown in Table 1. Total RNA was extracted from frozen intestine samples using an animal total RNA isolation kit (Foregene, Chengdu, China) according to the manufacturer's instructions.
The integrity and quality of the RNA were assessed using 1% agarose gel electrophoresis. The RNA concentration and purity were spectrophotometrically determined at 260/280 nm. Complementary DNA (cDNA) was synthesized using the RR047A kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. The polymerase chain reaction (PCR) was performed using a CFX96 (Bio-Rad, Hercules, CA, United States) according to the manufacturer's instructions. Reactions were performed in a 10-µl mixture made up of 1 µl of diluted cDNA, 5 µl of SYBR green master, 0.5 µl of forward primer, 0.5 µl of reverse primer, and 3 µl of PCR-grade water. The PCR program was 95 • C for 1 min and 40 repeated cycles of 95 • C for 10 s, and the appropriate melting temperature was 30 s. The melting curve revealed a single peak for each PCR product. Relative mRNA expression was calculated using the 2 − Ct method with the following formula:
DNA Extraction and Purification
Fifteen individuals of hybrid F1 from each group of four batches were randomly selected and humanely euthanized with MS-222. Intestinal fecal matter from five sturgeons was combined to make a pooled sample with three pooled samples per group. To ensure the efficiency of DNA extraction, 0.5 g of feces in each pooled sample was considered necessary. All intestinal content samples were sent to Shanghai Majorbio Biopharm Technology Co., Ltd. (Shanghai, China) for genomic DNA extraction. The DNA was extracted from the intestinal feces using the Bacterial DNA Isolation Kit (DE-05311, Foregene Company, Limited, China) and Plant DNA Isolation Kit (DE-06111, Foregene Company, Limited, China), according to the manufacturer's instructions. DNA concentration and quality were checked using a NanoDrop2000 (Thermo Fisher, Scotts Valley, CA, United States). The genomic DNA quality was assessed by 1% agarose gel electrophoresis. The V3-V4 region of the 16S rRNA gene was amplified by PCR with the primers proposed by Mori et al. (2014). Bacterial primer information: forward primer 338F: 5 -ACTCCTACGGGAGGCAGCAG-3 and reverse primer 806R: 5 -GGACTACHVGGGTWTCTAAT-3 . The ITS1 (internal transcribed spacer) region of the fungi rRNA gene was amplified by PCR with the primers proposed by Adams et al. (2013). Fungal primer information: forward primer ITS1F: 5 -CTTGGTCATTTAGAGGAAGTAA-3 and reverse primer ITS2R: 5 -GCTGCGTTCTTCATCGATGC-3 . The PCR products were detected by 2% agarose gel electrophoresis and purified with the AxyPrep DNA Gel Extraction Kit (Axygen, Corning, NY, United States), quantified using the QuantiFluorTM-ST Blue Fluorescence System (Promega, Beijing, China), and subjected to next-generation sequencing.
Sequencing and Quality Control
Sequencing of the 16S and ITS rDNA was performed on an Illumina Miseq PE300 platform (Illumina, San Diego, CA, United States) by Meiji Bioinformatics Technology Co., Ltd. (Shanghai, China). Two libraries were constructed for the V3-V4 and ITS1 amplicons. The paired-end (PE) sequencing was performed on the MiSeq system. The sequencing data were uploaded to the Sequence Read Archive at the National Center for Biotechnology Information (accession numbers PRJNA739968 and PRJNA738812). Based on the overlapping PE reads, pairend double-ended sequences were merged into single sequences using Flash software (Version 1.2.11). Raw fastq files were demultiplexed and quality-filtered by removing those that were shorter than 50 bp and greater than 10 bp in libraries or had ambiguous nucleotides that constituted over 20% of the sequence. According to the overlap of PE reads, the PE reads were merged into a sequence, and the minimum overlap length was 10 bp. The maximum allowable mismatch ratio in the overlapping region of Superscript a and b denote statistically significant differences among groups (p < 0.05).
the spliced sequences was 0.2, and any unmatched sequences were screened. Samples were distinguished according to the barcodes and primers at the beginning and end of their sequences, and the sequence direction was adjusted. The allowed number of mismatches in the barcode was zero, and the maximum allowable number of primer mismatches was 2. Non-repetitive sequences were extracted from the optimized sequences to reduce the number of redundant calculations in the middle of analysis. 1 All single sequences without repetitions were removed. 2 Operational taxonomic unit (OTU) clustering of non-repetitive sequences excluding single sequences was conducted according to 97% similarity. Chimeras were removed in the clustering process, and representative OTU sequences were obtained. All optimized sequences were mapped to the OTU representative sequences, and sequences that shared more than 97% similarity with the representative sequence were used to generate the OTU table. To obtain the species classification information corresponding to each OTU, the RDP classifier Bayesian algorithm was used to perform taxonomic analysis on the 97% similar level of OTU representative sequences. Each OTU was compared with the 16S rRNA database (Silva) and ITS rRNA database (UNITE) using BLAST analysis to obtain species classification information. In all sample analyses, species with relative abundance rates <0.01 were classified as "others."
Intestinal Microbiota Analysis
Species observed (Sobs) were used to evaluate actual richness, the Shannon and Simpson indexes were used to assess microbiota diversity, the Ace and Chao indexes were used to assess microbiota richness. The Venn diagram was prepared based on the alpha diversity analysis performed in Mothur. 3 The R language (version 3.3.1) was used to make the microbiota. Bar diagrams were used to visualize the dominant genus of 1 http://drive5.com/usearch/manual/dereplication.html 2 http://drive5.com/usearch/manual/singletons.html 3 https://www.mothur.org/wiki/Download_mothur each sample at the taxonomic level and the relative abundance (proportion) of each dominant genus in the sample. A beta diversity analysis was used to compare species diversity between different temperature groups. The abundances of the genus and the number of genera in each sample were counted. A visual circle diagram was used to reflect the connections among samples and the genus. The top 50 most abundant genera were used to visually study the microbiota composition using the heat map visualization method.
Statistical Analysis
All data were shown as the mean ± SD and assessed for homogeneity of variance. The IBM SPSS version 27.0 Statistics software (IBM Corp., Armonk, NY, United States) was used for the statistical analyses. Indicators related to heat stress were analyzed using one-way ANOVAs, and t-tests and differences among the two temperature groups at the same time point as well as the differences within groups at different time points were analyzed. Duncan's multiple comparison tests were used to compare means. The microbial diversity analysis database and software are shown in Supplementary Table 1. A normality test was conducted before the evaluation of the variables between the groups of the microbiota. The p-value was corrected by multiple hypothesis tests using the BH method (screening threshold: false discovery rate (FDR) <0.1, FDR = E(V/R), Abs (log 2 fold change) ≥2). p < 0.05 and p < 0.01 were considered significant and extremely significant, respectively.
RESULTS
The Effects of Heat Stress on Sturgeon Feeding, Survival Rate, and Physiological Indicators As the water temperature increased, the dissolved oxygen in the tank significantly decreased in the three temperature groups ( Figure 1C). Compared with the control and G1 groups, the fish in the G2 group had a significantly lower feeding rate ( Figure 1D). The cumulative survival rate of the control group was significantly higher than that of both treatment groups, and it was higher in G1 than G2 (Figure 1E). The final survival rates of the control group were 86.2%, G1 group 46.7%, and G2 group 31.4% survival. Plasma biochemical indexes have been widely used to assess the physiological function in animals (Lei and Xiu-Mei, 2004). In this study, the ALB, ALKP, CHOL, GLOB, GLU, LIPA, PHOS, and TP were all higher in the highest temperature group. The LPS, ALKP, and PHOS can be used to specifically assess the physiological function of the digestive system, and their plasma levels increased significantly with increasing water temperature ( Table 2). Overall, there was a significant decrease in feeding rate at the higher temperature, and it was speculated that the sturgeons' digestion was impaired.
Heat Stress Causes Enteritis of Valve Intestine in Sturgeons
Anatomical results showed that the valve intestine in the sturgeons of group G2 had dilated due to substantial amounts of gas, despite being devoid of food, and the valve intestinal walls had high transparency (Figure 2A). To further explore the effect of heat stress on the gastrointestinal tract, pathological changes were evaluated. Pathological lesions occurred in the valve intestine and were most serious in the G2 group (Figures 2C,D). Mucosal epithelial cells showed marked signs of necrosis, and shedding cells were seen in the lumen of the valve intestine accompanied by a large amount of inflammatory cell infiltration ( Figure 2B). The ultrastructural changes showed that heat stress had led to an enlargement of the mitochondria and endoplasmic reticulum, solidified chromatin, and blurring of the membrane boundary of the valvular intestinal epithelial cells (Figures 2B,E). The valve intestine of sturgeons in the elevated temperature group showed enteritis symptoms. No obvious lesions were present in the stomach or duodenum in any group, and no lesions were present in the valve intestinal of the control group.
Heat Stress Disturbs the Intestinal Microbiota Diversity of Sturgeons
To further examine the relationship between intestinal damage and intestinal microbiota in sturgeons at high water temperatures, the compositions and changes of the bacterial and fungal microbiota of the intestinal feces were analyzed. Through optimizing sequencing results and statistical analysis, a total of 479,104 effective bacterial and 611,088 effective fungal DNA sequences were obtained from nine intestinal samples, each sample representing the intestinal feces of five fish. High-quality reads were clustered based on a >97% sequence identity into 45,785 bacterial and 56,847 fungal OTUs. Rarefaction curves showed that these OTUs represented sufficient coverage and accurately reflected the bacterial and fungal compositions (Supplementary Figures 1A,B). Alpha diversity was used to evaluate the diversity of bacteria and fungi in the intestines of sturgeons. Almost all bacterial and fungal diversity indexes showed upward trends as the water temperature increased (Table 3).
Explosive Increase of Thermophilic Microbiota Triggers Intestinal Microbiota Disorders
The species, quantities, and proportions of intestinal microbiota were used to directly observe the differences in the microbiota of different treatment groups (Valdes et al., 2018;Aggeletopoulou et al., 2019). The Venn diagram illustrated that the number of OTUs of both bacteria and fungi was higher in the elevated temperature treatment groups than in the control group (Figures 3A,C). The microbiota composition analysis revealed that the bacterial microbiota of the control and heat treatment groups had significantly different dominant genus composition ratios. In the control group, Pseudomonas (51.36%) and Clostridium sensu stricto 1 (29.99%) were the dominant genera, whereas the proportion of dominant bacterial genus in the G2 group has changed significantly. The bacteria Cetobacterium (29.36%), Plesiomonas (18.24%), and Aeromonas (13.29%) all exhibited significant increases in abundance, while the abundances of Pseudomonas (13.28%) and C. sensu stricto 1 (4.58%) declined ( Figure 3B). The increased diversity of fungi is reflected in the non-dominant genus, which increased significantly in the G2 group, reflected in the abundance of Rhodotorula (0.48 to 2.87%) and Cladosporium (0.94 to 1.79%). As the water temperatures rose, the abundances of the fungi Cutaneotrichosporon (57.17 to 43.34%), Ascomycota (8.89 to 1.16%), and Aspergillus (5.51 to 0.22%) were significantly reduced ( Figure 3D).
The visual circle diagram was used to reflect the distribution ratios of each group of the dominant genus in the samples, as well as the distribution ratios of each dominant genus in separate groups. The top eight genera (others) in the dominant ratios were analyzed. The relative abundance values observed for each genus in the bacterial microbiota in the control group were Pseudomonas (45.00%), Cetobacterium (3.50%), C. sensu stricto 1 (84.00%), Rhodococcus (22.00%), Plesiomonas (0.28%), Aeromonas (0.33%), Turicibacter (0.02%), and Peptostreptococcaceae (0.13%). The proportions of Cetobacterium (60.00%), Plesiomonas (98.00%), Aeromonas (99.63%), and Turicibacter (42.00%) increased significantly in G2. In contrast, the ratio of Pseudomonas (12.00%) and C. sensu stricto 1 (13.00%) were significantly reduced by heat stress (Figure 3E). A quantitative analysis of the bacteria whose abundance had increased significantly showed that the average abundance value in the control, G1, and G2 were as follows: Cetobacterium 744,8,483 and 14,201,respectively;Plesiomonas shigelloides 26,150,and 8,332,respectively; and Aeromonas veronii 17, 1, and 6,081, respectively. The relative abundances of the fungal genera in the control group were Cutaneotrichosporon (42.00%), Rhodotorula (5.20%), Cladosporium (12.00%), Aspergillus (76.00%), Candida (54.00%), Cyberlindnera (20.00%), Fusarium (15.00%), and Engyodontium (0.13%). The proportions of Aspergillus and Candida decreased in G2, but the proportions of Rhodotorula and Cladosporium increased (Figure 3F). To better illustrate the change in genus abundances within the intestinal microbiota, the top 50 most abundant genera were selected to make a heat map. The horizontal abundance of the bacterial population between the G1 and control groups showed an intuitive dynamic change trend. Mycoplasma and Brevinema decreased significantly with increasing water temperature, while Plesiomonas, Aeromonas, Faecalibacterium, and Turicibacter gradually increased in abundance with increasing temperature (Supplementary Figure 2A). In the fungal microbiota, half of the population represented by Alternaria and Talaromyces Data are presented as mean ± SD (n = 9). Different lowercase superscript letters indicate significant differences between treatments (p < 0.05).
increased in group G2 compared with the control and G1 groups, but Xerochrysium, Penicillium, Xeromyces, and Aspergillus all decreased in group G2 (Supplementary Figure 2B). Principal component analysis (PCA) was used to perform a simplified analysis of the data set, and it showed that heat stress caused a significant difference between the group control and the G2 in bacterial ( Figure 4A) but in fungal microbiota, and the control and G2 groups had a sample overlap ( Figure 4B).
Heat Stress Suppresses the Digestive and Antioxidant Function of the Valve Intestine in Sturgeons
To examine the functional changes in the valve intestine, the digestive ability and antioxidant activity were determined in the valve intestine and in the plasma by measuring CHY, α-AMS, LPS, CAT, POD, and GPH-PX. The activity of CHY, α-AMS, and LPS in the intestines and POD and CAT activity in the plasma decreased with increasing water temperature (Figures 5A-C,F,H). However, the GXH-PX activity showed an opposite trend ( Figure 5E). The CHY, POD, and CAT activities were significantly lower in the elevated temperature treatment groups G1 and G2 (Figures 5A,F,H: p < 0.05). In addition, the CAT activity in valve intestine and GXH-PX activity in serum did not change significantly (Figures 5D,G). The results suggested that the decreased activity of digestive and antioxidant enzymes in the valve intestine may be ascribed to heat stress.
Reduced Thermal Tolerance and Repair Ability of Valve Intestine
The heat shock protein family genes Hsp60, Hsp70, and Grp75 and transforming growth factor Tgf-β were used to assess the stress status of the valve intestinal tissue. The analysis showed that Grp75 expression was significantly lower in the elevated heat treatments (G1) (Figure 6C) (p < 0.05). Of the remaining genes, Hsp60 and Tgf-β showed the same trends, and Tgf-β transcription was significantly higher in the G2 group (Figures 6A,D). However, the expression of Hsp70 did not change ( Figure 6B). Most of these results indicated decreases in thermal tolerance and repair capacity of the valve intestine at elevated temperatures.
DISCUSSION
Heat stress reduces the availability of suitable habitats for cold-water fish in freshwater environments and impacts their physiological and metabolic processes (Larnier et al., 2010;Schram et al., 2013;Huang and Wang, 2018;, directly affecting the health and biological function of cold-water fish (Miegel et al., 2010;Larios Soriano et al., 2018). The normal function of the intestine requires the participation of a healthy intestinal microbiota (Larios Soriano et al., 2018). Previously, high water temperatures have been reported to affect the intestinal microbiota of Salmo salar more than their diet (Neuman et al., 2016). Within the microbiota, bacteria and fungi play important roles in promoting food digestion, nutrient absorption, and the immune system (Yap and Marino, 2018). Under suitable water temperatures, beneficial microbiota can flourish, and high relative abundances of beneficial microbes can be maintained, helping to maintain the host's metabolic capacity and health (Dehler et al., 2017). In this study, the number of species and abundance of thermophilic bacteria and fungi were elevated in sturgeons exposed to high water temperatures, which was consistent with the results of the study by Soriano et al. on Yellowtail Kingfish in 2018, which showed that increases in ambient temperature increased the diversity of intestinal microbiota. The bacterial PCA in this study showed that there was a significant difference in composition between the control and G2 species. However, the composition of fungi of samples 1 and 2 was like that of G2, which may be due to the individual differences of sturgeons under heat stress and the low sensitivity of most fungi to temperature increase. This study saw an explosive growth of thermophilic bacteria and the severe decline of psychrophilic bacteria during heat stress, which significantly changed the composition of the microbiota. Previous studies reported that elevated temperatures had led to a significant increase in abundance of certain bacteria, such as Plesiomonas and Aeromonas, which had been found to have some degree of pathogenicity in Hypophthalmichthys molitrix and Lateolabrax maculatus (Behera et al., 2018;Wang et al., 2021). In the present study, the abundance and proportions of the thermophilic bacteria Plesiomonas and Aeromonas increased after heat stress. Studies have shown that outbreaks of pathogenic bacteria caused by high water temperatures can invade the blood from the intestine and lead to severe enteritis (Kanai et al., 2006;Chen et al., 2020). In this study, the relative abundance of Plesiomonas, which is a common aquatic pathogen that is speculated to be responsible for enteritis in sturgeons, was higher in G2 (Behera et al., 2018;Gong et al., 2019;. However, the abundance of Plesiomonas only increased significantly in two samples of the G2 group, which may be due to host differences and the low basic quantification of Plesiomonas. Aiming at the phenomena occurring in the valve intestine, Huang et al. (2020) speculated that Clostridium was the main cause of excess gas production. Based on the changes in genera compositions and abundances in the high-temperature group (G2), Cetobacterium and Aeromonas were suspected to be the main causes of valve intestine flatulence in this study (Wiegel et al., 2006;Ma et al., 2009). Fungi with increased abundance after heat stress, such as Rhodotorula, are not pathogenic in research reports (Raggi et al., 2014;Boguslawska-Was et al., 2019), and Cutaneotrichosporon has no pathogenicity studies. Therefore, we can deduce that it was the large proliferation of gas-producing bacteria caused by temperature elevation that induced the flatulence, decreased food intake, and no feces in the valve intestine of sturgeons.
In this study, no feces was present in the intestines of the high-temperature group, and a large number of pathogenic bacteria may attack the host's intestinal mucosa and cause serious damage to the intestinal tissue (Desai et al., 2016). The histopathological results of the sturgeon intestine in group G2 FIGURE 6 | Thermal tolerance capacity of sturgeon in different groups. Expression of heat shock proteins (A-C) and transforming growth factor (D) mRNA in sturgeon from different groups. ( * ) Indicates significant differences between groups (p < 0.05).
were similar to the enteritis symptoms reported by Huang et al. (2020), so it was speculated that heat stress caused sturgeon enteritis. Research has shown that environmental temperatures may influence physiological metabolic processes in fish by driving dramatic changes in the composition and abundance of intestinal microbiota (Kuz'mina et al., 2012(Kuz'mina et al., , 2019 and may inhibit the secretion of digestive enzymes and damage the structure of digestive enzymes (Ahmadifar et al., 2020). Clostridiaceae has been recognized as a beneficial bacteria that is involved in the breakdown of carbohydrates and proteins (Kim et al., 2008;Wuest et al., 2011), but the high water temperatures in this study inhibited their growth and led to a significant decrease in C. sensu stricto 1 abundance. Studies have indicated that Trichosporonaceae promotes the digestion and absorption capacities of hosts by secreting proteinases (Aliyu et al., 2020), but in this study, the abundance of Cutaneotrichosporon in the intestinal microbiota gradually decreased under high water temperatures, although a high metabolic rate was maintained. The transport and repair ability of proteins was reduced, and the proliferation of intestinal epithelial cells and vascular endothelial cells was inhibited.
Overall, the feed intake, the ability of digestion, and absorption of sturgeons were significantly inhibited in sturgeons, which was attributed to the damage of the intestinal structure, heat tolerance reduction, and reduced repair ability. Consequently, the sturgeons' energy supply was blocked, and microbial consumption was disordered, which inevitably posed a threat to the growth and survival of wild and cultured sturgeons. The absence or proliferation of intestinal microbiota in fish may lead to impaired physiological functions, such as intestinal epithelial cell dysfunction, compromised nutrient absorption, and metabolism . Therefore, a series of preventive measures will be indispensable in the protection and breeding of sturgeons. First and foremost, stable habitats with suitable water temperatures especially in summer play a vital role in the conservation of wild endangered sturgeons and breeding of farmed ones. Regular monitoring of both water temperature and the sturgeons' behavior is needed during farm management. Water cooling should be undertaken as soon as the water temperature in ponds rises dramatically to protect physiological functions and the maintenance of intestinal microbiota. In summer, the regular addition of endogenous probiotics such as Bacillus spp. and prebiotics to the diet or aquaculture water may protect the sturgeon intestines from the negative effects of heat stress (Geraylou et al., 2013). Natural extracts including resveratrol and arabinoxylan-oligosaccharides can also be added to the diet to enhance the anti-oxidation and antibacterial abilities of the sturgeon intestines and improve the growth performance (Geraylou et al., 2012;Liu et al., 2018;Zheng et al., 2018;Farsani et al., 2021). On the whole, effective and timely precaution can be essential to the resistance to heat stress for the protection and breeding of sturgeons and other cold-water fishes.
CONCLUSION
This study demonstrated that heat stress triggered the disturbance of the intestinal microbiota in A. baerii ♀ × A. schrenckii ♂ hybrid F1. The explosive increase of thermophilic microbiota and pathogenic bacteria genera including Plesiomonas, Cetobacterium, and Aeromonas may be associated with the development of enteritis. This was followed by the serious inhibition of intestinal digestion and antioxidant function. Simultaneously, heat stress reduced the thermal tolerance and weakened the repair ability of the valve intestine in sturgeons. The present work will be helpful for strategies making to enhance the resistance to thermal stress for wild and cultured sturgeons.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
ETHICS STATEMENT
The animal study was reviewed and approved by Animal Care and Use Committee of Sichuan Agricultural University Sichuan Agricultural University.
AUTHOR CONTRIBUTIONS
SY, CZ, and WX contributed to the data detection, conception, and design of the study. DL organized the database and data analysis. YF performed the statistical analysis. SY and CZ wrote the draft of the manuscript. JW, WL, and XD wrote sections of the manuscript. ZD and XH were responsible for reviewing and submitting articles. All authors contributed to manuscript revision and read and approved the submitted version. | 2022-03-07T14:22:55.565Z | 2022-03-07T00:00:00.000 | {
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197676763 | pes2o/s2orc | v3-fos-license | Inguinal Hernia: A New (Not Anatomical) Classification
Inguinal hernia is one of the most frequently found surgical problems, accounting for about 70-75 per cent of all hernia operations. Inguinal hernia represents a social disease, with considerable management costs. All classifications of inguinal hernia have something of arbitrary and artificial, and unfortunately are based on anatomic and functional criteria. Moreover, single hernia defect can be classified only during the operation and not in a preoperative setting. The aim of this study has been to evaluate the operative times and consequently identify factors that affect the surgical time. In this way we hope to create a new classification useful to standardize the operative time management. From February 2012 to June 2013, in the Day Surgery Unit of Campus Bio-Medico University of Rome, 110 consecutive patients were enrolled which underwent to inguinal hernioplasty, and they have been observed by the same surgical team. We evaluated clinical parameters (age, sex, BMI, hernia size defect, reducibility, primitive or recurrent hernia, previous hernia surgery) and compared them with surgical times. Data analysis shows a statistically significant relationship between reducibility, recurrent hernia, male gender, BMI and surgical times. This study confirms that an optimal clinical patient evaluation should always be the first step to an effective organizational choice and it allows realistic predictions about the duration of inguinal hernioplasty.
Introduction
Inguinal hernia is one of the most frequently found surgical problems, accounting for about 70-75 per cent of all hernia operations [1]. Approximately, more than 20 million patients undergo inguinal hernia repair [2]. The incidence of inguinal hernia repair is 10 per 100000 population in the UK and 28 per 100 000 in the USA [3].
Twenty-seven percent of men and three percent of women will develop an inguinal hernia during their lifetime. Inguinal hernia represents a social disease, with considerable management costs [4].
Worldwide men and women have a 27% and 3% risk to develop an inguinal hernia during their life [5].
According to data from the National Center for Health Statistics, recent studies have estimated between 600 and 770 surgical repairs of inguinal hernia annually and, mostly were performed on outpatients [6].
It's unknown the real recurrence rate after an inguinal hernia repair, but approximately a 10-15% of patients require a reoperation [7].
Nowadays is increasingly widespread the concept of a tailored surgical operation for each patient, including a minimal recovery, and low invasiveness, cost, recurrence rate, and post-operative pain [8].
The first inguinal hernia repair were performed at the end of the sixteenth century and it provided the reduction and resection of the hernia sac, and the reinforcement of the inguinal canal posterior wall by approximating its muscular and fascial components [2].
Currently, groin hernia treatment is not standardized, in fact in the Literature have been reported three possible choice: open, laparoscopic and robotic approach [2].
Anyway, the open approach represent the treatment of choice for the majority of surgeons, due to low costs, low length of stay (outpatients setting or day-surgery recovery), ease of execution, and minimal invasiveness. Since 1804, inguinal hernias were traditionally classified as indirect, direct, and femoral by Cooper [12]. Since then, many classifications have been proposed. In particular, in 1987, Lichtenstein reported a 6000 cases series including his personal classification system, including three categories: indirect, direct and femoral [13]. The most recent classification was published in 1999 by Zollinger [14,15].
Unfortunately, all classifications of inguinal hernia have something of arbitrary and artificial, and are based on anatomic and functional criteria [16]. Moreover, single hernia defect can be classified only during the operation and not in a preoperative setting.
In this study we reviewed the main classifications of inguinal hernia in scientific literature.
We looked at the issue from a different perspective: is it possible, using pre-operative evaluation of patients, to define clinical criteria that help the surgeon to estimate the complexity of the single inguinal hernia repair and consequently, times of surgery? A functional pre-operative evaluation would allow a better surgery scheduling and increase the efficiency of operating room.
The aim of this study has been to evaluate the operative times and consequently identify factors that affect the surgical time. In this way we hope to create a new classification useful to standardize the operative time management.
Material and Methods
From February 2012 to June 2013 we have consecutive enrolled 110 patients, aged between 18 and 84 years, 10 females and 100 males, affected with inguinal hernia and candidates to monolateral inguinal hernioplasty procedure.
In 98% of cases the inguinal hernioplasty was performed with a sutureless and tension free open technique. We proceed to a clinical and preoperative evaluation of each patient, according to criteria such as: age, Body Mass Index (BMI), sex, specifying whether right or left, size of the hernia (then -Width -Length -Area classifying as W1 hernias of maximum diameter less than 4 cm, W2 hernias with a maximum diameter between 4 and 10 cm and W3 large hernias, that is, with diameter greater than 10 cm), size of the hernia door in cm, reducibility (indicating as "1" spontaneously reducible hernia, for example in supine position, as "2" hernia only manually reducible, as "3" an incarcerated hernia), primary or recurrent hernia, any mesh used in case of relapse, previous abdominal surgery.
To avoid bias of concordance, clinical evaluations and operation on patients, has been performed by the same surgical team, using the same surgical technique and local anesthesia. Data regarding operative time, has been collected in a database and statistical analysis to identify a possible relationship between these criteria and surgical times (from skin incision to skin closure) has been performed, either individually or in combination. Student's T test was used to evaluate the statistical significance as parametric measure of these relations.
Results
Patient's characteristics are reported in Table 1. Table 1 Characteristics and clinical parameters of patients with inguinal hernia, that we correlated with surgical time. The mean surgical time required to perform the hernioplasty procedure was 63.78 minutes, with a minimum time of 24 minutes and a maximum time of 108 minutes.
We analyzed all clinical parameters in our patients, in order to know how they affect the surgical time, as reported in Table 2. Table 2 Parameters that affect surgical time. Reducibility: completely reducible hernia and notcompletely reducible hernia. A completely reducible hernia is associated with shorter operating times than not-completely reducible hernia: 50.73 minutes (CI − confidence interval 95% 44.3 -57.13 minutes) in the first group versus 69.70 in the second. A statistically significant difference has been found between two groups using the Student t test for unpaired data (p < 0.001) (Figure 1). The irreducibility is therefore definitely as a variable that increases the mean surgical times.
Recurrent hernia: it needs on average 20 ± 5 minutes longer than the primary hernia (p < 0.05) (Figure 2). Sex: the female sex has been found associated with shorter mean surgical times (-13 minutes) compared to the male sex. The difference has been statistical significant (p <0.05) (Figure 3). BMI: we have divided patients into two groups on the basis of cut-off classically used to distinguish normal weight subjects from the overweight subjects (BMI between 18 and 24.9 = normal weight; BMI above 25 = overweight). In overweight/obese patients we observed longer surgical times than in normal weight patients, quantifiable in 14 ± 4 minutes (p < 0.001) (Figure 4).
Discussion
In Day Surgery, we perform an average of ten surgical operations of unilateral inguinal hernia repair in two operating sessions every week. A single operative session lasts 7 hours, from 7 am to 2 pm. The mean time of each procedure is about 67 minutes and the turnover between two procedures is 10 minutes. Accordingly, five operations can be performed for each session, but many factors can influence surgical times and daily schedule. This inaccuracy in predicting case durations can result in three possible scenarios with obvious consequences on the operating room efficiency and on patient care: a) overutilization of Operating Room (OR); b) underutilization of OR, with excess staffing costs due to OR allocation not being based on maximizing OR Efficiency; c) one or more case cancellation, with consequent patient discomfort.
No classification is useful for preoperative planning considering surgical times. Indeed, while a novel direct hernia can be repaired in few minutes, a recurrent indirect inguinal hernia may require an hour.
From our study, we can highlight a statistically significant relationship between the surgical times and clinical parameters, such as the patient's sex, hernia reducibility, recurrent hernia and BMI. The classical distinction of inguinal hernias between direct and oblique was excluded from the criteria because clinical examination is often not sufficient to discriminate between the two different types of defect.
Based on these parameters, we can identify three patient groups, as shown in Table 3. Table 3 The our new classification of patients in 3 groups, based on predictive clinical parameters of surgical times. Conditions such as female sex, primitive and completely reducible hernia, BMI minor than 25, lead to a surgical time equal to or less than 63 minutes (group A).
Male sex, not completely reducible hernia, and a BMI greater than 25, determine time longer than the average time for the intervention of inguinal hernioplasty of 10-25 minutes (expected time 64-88 minutes -group B).
In patients with recurrent hernia or not reducible hernia (which constitutes 60% of the sample) we recorded average surgical time of more than 88 minutes (group C).
Estimated times are important for simulation models and for decision analysis based on such simulations.
Based on the aforementioned patient classification we will study a new OR organization, increasing efficiency.
Our results has been obtained by working with a fixed operating room team on consecutive similar cases as demonstrated by several studies [17,18].
This experience can yield a new, not anatomical, classification of patients at the moment of the surgical visit hence facilitating the OR management where operating efficiency co-exists with the availability of time necessary and sufficient to perform the surgery "to perfection".
Conclusions
The aim of our study was to evaluate the operative times and consequently identify factors that affect the surgical time. As this study confirms, an optimal clinical patient evaluation should always be the first step to an effective organizational choice and it allows realistic predictions about the duration of inguinal hernioplasty. To know, also approximately, the duration of a surgical operation allows to reduce operating room costs and a minor time lose, ensuring more surgeries for each operative session.
This new classification can easily be adopted by every surgeon worldwide, ensuring a tailored operating session.
Further studies will be needed to enforce the effectiveness of this organizational model. | 2019-07-26T07:23:32.284Z | 2019-06-24T00:00:00.000 | {
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234653138 | pes2o/s2orc | v3-fos-license | Study of Clinicobacteriological Profile of Neonatal Sepsis Patients Admitted In Tertiary Care Hospital in Telangana
Original Research Article Background: Septicemia in new born is of major public health concern, though recent medical advances have improved neonatal care, there are still many challenges remaining in the diagnosis and management of neonatal infection. Immature immune system and invasive life support make the premature neonates susceptible to infections. The diagnosis is complicated especially by the presence of noninfectious conditions. Objective: To study the Clinical and Bacteriological profile and antibiotic susceptibility pattern of septicemic neonates in the microbiology department, Niloufer hospital and find the significant risk factors leading to cause septicemia. Methods: Retrospective study was carried out in the Department of Microbiology at Niloufer Hospital, Red hills between January 2019 to Nov 2019.737 clinically suspected samples were included in the study, were processed. Identification and antibiogram of the isolates were done as per standard procedures. Clinical symptoms, Laboratory Parameters, Risk factors were statistical analysed, pvalue < 0.05 was considered significant. Results: Among 737 samples, 135 isolates identified and grouped.Gram positive organism 98 (72%) out of which Coagulase Negative Staphylococcus 93(68.9%). CoNS causing Proven Sepsis were 66 (48.9%). Among Gram Negative Organism Klebsiella pneumonia (48.6%) was commonest. Elevated CRP levels was significant to categorise CoNS isolate. Neutropenia and Thrombocytopeniawere significant in Gram negative sepsis. All the clinical symptoms and Risk factors were significant. Conclusion: Low birth weight and premature neonates are the greatest risk factors for the neonatal sepsis. Immature host defense mechanism and prolong use of CentralVenous catheters, mechanical ventilation, parenteral nutrition has led to the increase risk of CoNS infection in Late onset sepsis.By updating the Antibiotic policy can decrease the usage of Catheters and early shifting to enteral feeding can decrease the chance of late onset sepsis.
INTRODUCTION
Sepsis in new born is of major public health concern. It is an extreme body response to invasion of blood stream by microorganism leading to systemic signs of infections. Newborn are at higher risk of developing sepsis leading to severe morbidity and mortality. India with nearly 6, 00,000new born deaths each year accounts for a quarter of the global burden of neonatal death (Unicef) [1]. Mortality due to septicemia has taken third portion after prematurity and perinatal asphyxia [2]. Sepsis occurring just after birth within 72hrs is termed as early onset due to maternal transmitted pathogens via genital tract during intrapartum period. Group Neonates become colonized by microorganisms present in environment with in first week of life. Sepsis caused by Coagulase negative staphylococcus (CoNS) is the major pathogen involved in LONS particularly at a low gestational age due to Microbiology improper development of immune system. CoNS infection in newborn are rarely fatal, but they cause significant morbidity and multiresistance to antibodics .Being a common inhabitant of skin and mucous membrane, risk of infection due to coagulase negative staphylococcus increase with the use of central venous catheter, mechanical ventilation , parenteral nutrition and with other invasive procedures at Neonatal intensive care units. Presence of CoNS organism in neonatal sepsis is of dilemma as can reflect contamination rather than true bacteremia. Clinical presentation of sepsis in neonates is not specific unlike in older patients [7][8][9][10]13]. Inorder to categorise the true cases of CoNS causing septicemia two positive blood cultures are required but in preterm neonates due to risk of multiple punctures , single positive blood culture and positive CRP (>10mg/l) considered as proven sepsis. Positive blood culture with negative CRP is considered as Contaminant [7] . WHO identified seven clinical signs-difficult feeding, convulsions, movement only when stimulated, respiratory rate >60/ min, severe chest indrawing and axillary temperature >37.5 0 c or < 35.5 0 c [3]. Perinatal antibiotic use, minimal blood volume sample for culture and repeated invasive procedures has made the diagnosis a big challenge [7].
Our aim is to study the clinicobacteriological profile of neonatal sepsis admitted at niloufer hospital, Redhills and to study the antibiotic resistance pattern to provide an effective early diagnosis for the clinicians to treat the patients.
METHODS
Retrospective study was conducted from January 2019 to November 2019 in the department of Microbiology, Niloufer hospital, Red hills. Sample size of 737 clinically suspected septicemic cases were included in the study. Neonates with congenital malformations, known chromosomal disorder were not included in the study. Premature neonates with low birth weight, risk factors like indwelling catheter, ventilation, parenteral nutrition developing sepsis after 72 hrs of their birth, were included in the study.
Sample collection
1ml of blood was collected in to 10ml of Tryptone soya broth and processed as per the protocol [5,6] and incubated for one week at 37 0 c and was checked for bacterial growth daily. Subcultures were done on Blood agar, Mconkey Agar after 24 hrs of incubation if no growth occured on plates, subsequent cultures done on 3 rd , 5th, 7 th , day. The growth bacterium was identified by colony morphology, gram stain and standard biochemical tests [5,6]. The Antibiotic susceptibility testing was performed by Kirby bauer disc diffusion method for the bacterial isolates, as per clinical and laboratory standard institute guidelines (CLSI). ATCC control strains were used accordingly as per standard procedures. Blood cultures positive for organism like corynebacterium, propionibacterium, penicillium and diphtheroids are considered as contaminants. Laboratory parameters like CRP, Hematologic indices (Thrombocytopenia, neutropenia) were also taken to distinguish CoNS isolation a pathogen or contaminant. The antibiotic discs (Himedia Co, Mumbai, India) and their concentrations per disc (ug) are: Ampicillin (10)
DATA ANALYSIS
All clinical symptoms, risk factors significant for causing sepsis were statistically analysed. The chi square test was used in assessing the association between variables.Statistical significance was defined as p<0.05.
RESULTS
There were 737 preterm infants with less body weight, 135 showed pathogenic organisms. The incidence of neonatal sepsis was 18.3 % among 737 blood samples enrolled in our study. Among the positive cases the bacteriological profile shows: CoNS was the most common pathogen followed by Klebsiella pneumonia 18 (13.33%). All the proven CoNS infection were sensitive to vancomycin, linezolid, Teicoplanin. 36(38.7%) CoNS isolate were resistant to cefoxitin. All gram negative bacteria were sensitive to imipenem, Amikacin, Gentamicin and Meropenem. Presence of catheter at the time of sepsis and complete perenteral nutrition in preterm babies were identified as independent risk factor for CONS-LONS. Prolong use of catheters; parenteral nutrition was associated with increased risk of late onset sepsis, thus removal of catheters with enteral feeding should be initiated.
CONCLUSION
Advancements in neonatal intensive care units and routine antenatal checkups have decreased the incidence of inborn sepsis but preterm deliveries and low birth weight with low immune response has increased the incidence of late onset sepsis. Bacteriologic profile will help in updating the antibiotic policy to provide better treatment to the patients. | 2020-12-07T05:44:27.478Z | 2020-11-09T00:00:00.000 | {
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119058081 | pes2o/s2orc | v3-fos-license | Possible d+id scenario in La_{2-x}Sr_{x}CuO_4 by point-contact measurements
We analyze the results of point-contact measurements in La_{2-x}Sr_{x}CuO_{4} (LSCO) previously reported as a clear evidence of the separation between gap and pseudogap in this copper oxide. Here we show that, in addition to this, the conductance curves of our point-contact junctions -- showing clear Andreev reflection features -- can be interpreted as supporting a nodeless d_{x^2-y^2}+id_{xy}-wave symmetry of the gap in LSCO. The results of our analysis, in particular the doping dependence of the subdominant d_{xy} gap component, are discussed and compared to the predictions of different theoretical models.
Introduction
In spite of the large number of experimental evidences and theoretical arguments supporting a pure d x 2 −y 2 symmetry of the order parameter in cuprates 1,2 , the possible existence of a subdominant component with different symmetry has also been deeply investigated. One of the reasons is that most of the experimental probes cannot really exclude the presence of a small additional component. Another reason is that some tunneling experiments along the ab plane of YBa 2 Cu 3 O 7−δ (YBCO) have shown a splitting of the zero-bias conductance peak (ZBCP) both in the presence 3,4,5,6 and in the absence 3,4,7 of a magnetic field. A possible explanation of this phenomenon stems from the idea that the ZBCP is due to zero-energy Andreev bound states at the surface 8,9 that experience a Doppler-like shift to finite energy in the presence of supercurrents. In the absence of a magnetic field, such a shift might be due to spontaneous supercurrents due to the breaking of the time-reversal symmetry. According to Fögelstrom et al. 10 a subdominant pairing interaction with smaller critical temperature can in fact appear at the surface of a d-wave superconductor, with a phase shift of π/2 with respect to the dominant one. This gives rise to spontaneous supercurrents and to a local breaking of the time-reversal symmetry.
An alternative picture has been emerging in the last years, in which an intrinsic instability of the d-wave superconductor toward a time-reversal breaking state is supposed, with no relation to surface effects. This picture is somehow based on the indications of a quantum critical point (QCP) in the proximity of optimal doping, obtained in Bi 2 Sr 2 CaCu 2 O 8+δ (BSCCO) by ARPES 11 . The hypothesis has been made that such a quantum critical point could mark the transition from a pure d-wave superconducting state to a time-reversal symmetry breaking state, such as In the present paper, we present a possible indication of a d + id scenario in La 2−x Sr x CuO 4 (LSCO) obtained by (re)analyzing the results of point-contact measurements in polycrystalline LSCO samples with various doping contents. These data were already reported in a previous paper 16 in a rather different groundwork, i.e. they were shown to evidence the separation between superconducting gap and pseudogap in underdoped LSCO.
Experimental details
We used La 2−x Sr x CuO 4 polycrystalline samples with various doping contents from strongly underdoped to slightly overdoped: x = 0.08, 0.10, 0.12, 0.13, 0.15 and 0.20. Details about the sample preparation and characterization are given elsewhere 16,17 . The critical temperatures, determined by means of magnetic (a.c. susceptibility) and transport (resistivity) measurements, resulted in good agreement with the standard T c vs x curve for LSCO 18 .
Point contacts were obtained by gently pressing sharp Au tips (whose endingpart diameter was always less than ∼ 2 µm) against the surface of the samples. We often obtained SN junctions with clear Andreev reflection characteristics. In some cases, the stability of the point contacts allowed us to follow the evolution of the conductance curves on heating the junction from 4.2 K up to the temperature T A c at which the dynamic conductance dI/dV was flat.
A discussion of the regime of current flow through our point contacts was already reported elsewhere 16 . Here let us just remind that we systematically rejected all the data sets showing an anomalous temperature and voltage dependence of the normal-state conductance (for example, for V > 20 mV) that usually indicate the presence of heating effects in the junction 19 . As a result, all the curves that have been used for the following analysis can reasonably be thought of as obtained in a regime of ballistic current flow through the junction, thus allowing us to perform spectroscopic measurements with a good energy resolution (< 1 meV). Fig. 1 reports the experimental conductance curves at low temperature (4.2 ÷ 5.6 K) for all the aforementioned doping contents, normalized to the normal-state conductance -so that they tend to unity at high positive (negative) voltage. The curves have been shifted vertically for clarity. Solid lines represent the best-fitting theoretical curves calculated by using the BTK model 20 generalized by Tanaka and Kashiwaya 9,21 with a d x 2 −y 2 +id xy symmetry of the order parameter. The details of the fitting procedure are reported elsewhere 17 . The fitting parameters are ∆ x 2 −y 2 and ∆ xy , Z (related to the height of the potential barrier) and the broadening parameter Γ that was always kept as small as possible.
Results and discussion
Even at a first glance, the fit appears rather good. Notice that the "dip" present in some curves, which is a fairly typical feature, cannot be fitted at all by the model, irrespective of the gap symmetry used. It must be said here that, as pre- viously reported 16 , various other symmetries were tried: s, d, s + id, extended s and anisotropic s, and none of these could give good results, especially when the temperature evolution of the curves was considered. Only the s + d symmetry was found to fit almost equally well the experimental data a , but its compatibility with the symmetry of the LSCO lattice at these doping levels is questionable 1,22 . The fit of the low-temperature conductance curves shown in Fig. 1 gives the doping dependence of the low-temperature gap components, ∆ x 2 −y 2 and ∆ xy , reported in Fig. 2 (solid circles and squares, respectively). The error affecting each gap value is rather small (about the size of the points) b . The amplitude of the gap, |∆| = ∆ 2 xy is also shown (solid triangles). It is clearly seen that the d xy component is present for all doping levels and is always smaller than the d x 2 −y 2 one -though representing a substantial part of the total amplitude. Neither ∆ Fig. 1. A comparison is made with the results of tunnel and ARPES measurements (from refs. [23] and [24], respectively), and with the standard Tc vs x curve (from ref. [18]) .
the tunneling gap (open squares) and the ARPES leading-edge shift (open circles).
Rather, a decreasing tendency is evident in the underdoped region. A comparison is also made with the standard curve of T c versus doping (thick solid line) 18 , which is strikingly similar to the ∆ xy (x) curve and, with less accuracy, to the |∆|(x) one. Notice that a strong suppression of both ∆ x 2 −y 2 and |∆| occurs at x = 1/8, where also T c is reduced, further indicating a close relationship between the Andreev gap and the critical temperature. Thus, the conclusion holds true that we drew in a previous paper 16 : Andreev reflection does measure the superconducting gap, as opposed to ARPES and tunnel spectrocopies that instead measure the pseudogap.
Further support to this assertion comes from the temperature dependence of the conductance curves of our junctions. In all cases, in fact, the Andreev-reflection features disappear at a temperature T A c close to or smaller than the bulk critical temperature measured by resistivity, with no evidence of persistence of the gap above T c . The values of T A c are reported for each doping level in Fig. 2 (open triangles). Fig. 3 shows, as an example, the temperature evolution of the curve for x = 0.20 already shown in Fig. 1, together with the d + id best-fitting curves obtained by keeping Z constant (Z = 0.135, which is the value at 4.2 K).
Fitting the normalized conductance curves at all temperatures allows us to obtain the temperature dependence of the two gap components, which is shown for the case x = 0.20 in Fig. 4. It is clearly seen that the d xy component is always smaller than the d x 2 −y 2 one, and that the thermal evolution of both components follows a very similar trend, rather different from a BCS curve. Notice that the critical temperature T c2 of the subdominant d xy component is smaller than T c . A very similar thermal evolution of the gap components is observed also for the remaining doping levels. Further details will be given in a more extended paper. −y 2 ) and subdominant (dxy) gap components on the temperature, obtained from the fit of the conductance curves shown in fig.3. Error bars indicate the range of values that give an acceptable fit when the remaining parameters are suitably adjusted (note that Z was fixed to its low-T value). Dashed lines are guides to the eye. It is well clear that the dxy component closes at a lower temperature. The same happens at all doping contents.
Conclusions
As far as the gap symmetry is concerned, our findings agree with some tunneling measurements in optimally-doped LSCO, that evidenced the absence of nodes in the gap 25 and also with previous Andreev reflection experiments 26 that were interpreted as supporting a mixed symmetry. Of course, the question whether the additional d xy component arises from surface effects or from a quantum phase transition cannot be addressed by our measurements. However, it must be said that the presence of the subdominant d xy pairing in the whole doping range analyzed, as well satt11˙did˙rev: submitted to World Scientific on March 22, 2022 as its dependence on the doping (see Fig. 2) disagree with the findings in YBCO films 4 . In that case, the spontaneous splitting of the zero bias in the tunneling conductance (proportional to the amplitude of the d xy component) was observed only above optimal doping, and turned out to increase monotonically with increasing doping. This behaviour was indeed used to argue for a quantum critical point near optimal doping, and was reproduced by some theoretical models predicting the stability of the d + id phase in the overdoped regime 14,15 . What our results say, instead, is that either the time-reversal symmetry breaking is a surface effect with no relationship to quantum criticality 10 (and perhaps related to doping only through the amplitude of the dominant gap component), or the quantum critical point is placed somewhere in the extreme underdoped or extreme overdoped region of the phase diagram. Further measurements in these two extreme regimes will possibly help in discriminating between these two possibilities. V.A.S. acknowledges the support from RFBR (project N. 02-02-17133). | 2019-04-14T02:00:31.752Z | 2002-07-17T00:00:00.000 | {
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52005576 | pes2o/s2orc | v3-fos-license | The Heidelberg Milestones Communication Approach (MCA) for patients with prognosis <12 months: protocol for a mixed-methods study including a randomized controlled trial
Background The care needs of patients with a limited prognosis (<12 months median) are complex and dynamic. Patients and caregivers must cope with many challenges, including physical symptoms and disabilities, uncertainty. and compromised self-efficacy. Healthcare is often characterized by disruptions in the transition between healthcare providers. The Milestones Communication Approach (MCA) is a structured, proactive, interprofessional concept that involves physicians and nurses and is aimed at providing coherent care across the disease trajectory. This study aims to evaluate these aspects of MCA: (1) the training of healthcare professionals, (2) implementation context and outcomes, (3) patient outcomes, and (4) effects on interprofessional collaboration. Methods/design A multiphase mixed-methods design will be used for the study. A total of 100 patients and 120 healthcare professionals in a specialized oncology hospital will be involved. The training outcomes will be documented using a questionnaire. Implementation context and outcomes will be explored through semi-structured interviews and written questionnaires with healthcare professionals and with the training participants and through a content analysis of patient files. Patient outcomes will be assessed in a pragmatic non-blinded randomized controlled trial and in qualitative interviews with patients and caregivers. Trial outcomes are supportive care needs (SCNS-SF34-G), quality of life (SeiQol and Fact-L), depression and anxiety symptoms (PHQ-4), and distress (Distress Thermometer). Qualitative semi-structured interviews on patients’ views will focus on shared decision-making, communication needs, feeling empathy, and further utilization of healthcare services. Interprofessional collaboration will be explored using the UWE-IP-D before the implementation of MCA (t0) and after 3 (t1), 9 (t2), and 12 (t3) months. Discussion Using guideline-concordant early palliative care, MCA aims to foster patient-centered communication with shared decision-making and facilitation of advance care planning including end-of-life decisions, thus increasing patient quality of life and decreasing aggressive medical care at the end of life. It is assumed that the communication skills training and interprofessional coaching will improve the communication behavior of healthcare providers and influence team communications and team processes. Trial registration German Clinical Trials Register, DRKS00013649 and DRKS00013469. Registered on 22 December 2017. Electronic supplementary material The online version of this article (10.1186/s13063-018-2814-1) contains supplementary material, which is available to authorized users.
(Continued from previous page)
Discussion: Using guideline-concordant early palliative care, MCA aims to foster patient-centered communication with shared decision-making and facilitation of advance care planning including end-of-life decisions, thus increasing patient quality of life and decreasing aggressive medical care at the end of life. It is assumed that the communication skills training and interprofessional coaching will improve the communication behavior of healthcare providers and influence team communications and team processes. Trial registration: German Clinical Trials Register, DRKS00013649 and DRKS00013469. Registered on 22 December 2017.
Background
The care of patients with a limited prognosis is complex and demanding. Moreover, the process of care is often interrupted by quick transitions between healthcare settings and providers, especially for cancer patients [1], leading to a lack of continuity in care. Throughout the disease trajectory, patients and caregivers must deal with receiving bad news at short intervals. The terminal character of a limited prognosis means that patients need to engage in advance care planning and end-of-life decision-making. Therefore, patients and their caregivers must deal with several dimensions of burden (physical, psychological, social, financial, and spiritual) [2].
The quality of care for patients with advanced cancer hinges, among other factors, on the communication skills of the healthcare professionals involved. Delivering bad news, discussing prognoses and possible disease trajectories (best case and worst case), and preparing others for the end of life are challenging communication topics and demand highly skilled professionals [3]. Yet, patients, caregivers, and the healthcare professionals themselves often perceive these skills to be insufficient [4,5].
The German National Cancer Plan [6] and other medical societies recommend that healthcare providers should improve their communication skills [6][7][8]. Indeed, different communication training programs have been developed and evaluated successfully for the medical curriculum [9,10]. Yet, most of these programs focus on basic communication skills in specific situations [11][12][13][14] without considering the whole disease trajectory. Furthermore, communication modules concerning palliative care have been developed but refer mainly to the transition to the best supportive care for the terminally ill [15]. The positive results of trials regarding the early integration of palliative care [16,17] additionally challenge patient-physician communications as they leave the oncologist with the task of addressing palliative care early in the course of disease.
These trials and others that evaluate the training of communication skills in oncology have shown multiple benefits: improvement of quality of life and advance care planning for patients, improvement in job satisfaction, and decrease of burden on professionals [12,13,[18][19][20][21]. Additionally, different studies on palliative care show that multi-professional approaches are more effective [16,[22][23][24].
For advanced lung cancer, forward-thinking communication has been described for the German setting [25]. This introduces standardized steps of communication at turning points of treatment, such as first disclosure of diagnosis, disease progression, and transition to best supportive care. Based on findings from interviews with patients and caregivers and focus groups with healthcare professionals, a structured longitudinal concept has been developed: the Heidelberg Milestones Communication Approach (MCA) [5,26,27].
MCA is a pro-active, interprofessional concept that involves physicians and nurses and is aimed at providing coherent care that integrates palliative care early and across the disease trajectory. It provides intervention-based communication to develop patient-centered care further through increasingly integrating patient preferences. Metastatic lung cancer has been used as a model disease in developing MCA. Not only is there a limited prognosis with a median survival of less than 12 months but it is also associated with a substantial existential uncertainty and a high symptom burden with detrimental impact on patients and caregivers [27,28]. However, institutional strategies for implementing longitudinally structured communication concepts such as MCA and knowledge about the effects of implementation are still lacking.
Many interventions in clinical and health services research fail to translate into practice and policy [29]. Implementation processes are complex, take a long time, and are cost intensive [30,31]. Therefore, the effects and implementation of an intervention need to be evaluated concurrently. Furthermore, implementations are influenced by individual health, professional factors, patient factors, professional interactions, incentives and resources, capacity for organizational change, as well as by social, political, and legal factors [32].
Aims and objective
This study aims to evaluate these aspects of MCA: 1. training of healthcare professionals 2. implementation context and outcomes 3. patient outcomes 4. effects on interprofessional collaboration.
Methods/design
Multiphase mixed-methods design Since several perspectives (patient, family caregiver, and healthcare professional) and different interventions (communication concept, training, and implementation) at different stages (development, implementation, and evaluation) are relevant, a mixed-methods design has been chosen [33][34][35]. Our aims contain complex multidimensional processes (social, cognitive, and cultural) and multiple stakeholders (patients, caregivers, and healthcare professionals). To evaluate outcomes, a randomized controlled trial (RCT) and interviews with patients and caregivers are planned. To evaluate processes and context, a combination of quantitative and qualitative methods will be applied [35]. The multiphase mixed-methods design allows a comprehensive understanding of the effects of MCA [34].
Phase 1: development of the Heidelberg MCA
Our previous studies in phase 1 included a qualitative exploration of patients' and caregivers' experiences over the disease trajectory and the assessment of healthcare professionals' views of a hypothetical structured and forward-thinking communication approach [5,26,27]. Based on these results, Heidelberg MCA was developed as a complex intervention: (a) to address the communication needs of patients and caregivers, (b) to improve continuity of care, (c) to improve individual quality of life of patients and their caregivers, (d) to foster shared decision-making including end-of-life decisions, and (e) to enhance communication competencies and team processes of the interprofessional oncology team.
In a second step, we led in-depth interviews with nurses and focus group interviews with physicians at our institution to explore the enablers and barriers of implementation as well as interprofessional collaboration concerning all four components of MCA. Based on the results of these interviews, MCA was adapted and now contains the following components (see Table 1): 1. Interprofessional (physician and nurse) communication training 2. Planned, structured nurse-physician-patient and caregiver conversations at four points within the disease trajectory, the so-called milestone conversations 3. Monthly follow-up sessions for outpatient and ambulatory patients and their caregivers (nurse) 4. Supportive materials (question-prompt-list, managing symptoms guidebook for patients and caregivers, and communication manual and memory cards for nurses and physicians) For a more detailed description of the intervention, see Additional file 1.
No harm is anticipated from the intervention. If any harm is identified, a referral to psycho-oncology services is possible.
The complete project consists of three phases: (1) further development, (2) implementation, and (3) evaluation. Phase 1 (further development) had a separate ethical approval (ethics committee University of Heidelberg S-139/2017) and is already completed. This study protocol is for phase 2 (implementation) and phase 3 (evaluation). Implementation and evaluation are based on the results of phase 1. During the implementation phase, communication training will be conducted and evaluated. Implementation of MCA will be adapted according to participant experiences. The evaluation phase includes a monocentric non-blinded RCT measuring the effects of MCA in patients with a limited prognosis. In a cohort study, the longitudinal effects on interprofessional collaboration of clinical staff will be observed. Experiences with the concept will be evaluated in qualitative semi-structured interviews with patients and their caregivers (see Table 2).
To implement MCA, it is important to understand how MCA works within a real-life clinical setting. This implementation implies working with healthcare professionals who will be affected by MCA and investigating the context of the intervention. A hospital context is not a fixed organizational structure but an unstable, unfolding process [36]. Thus, implementation of MCA will be conducted using a plan-do-check-act (PDCA) cycle [37]. If necessary, the PDCA cycle will be used several times. This approach ensures that the adaptability, usability, and feasibility of MCA are incorporated [32].
Setting
The project will be conducted at the Department of Thoracic Oncology, University Hospital Heidelberg, which is one of the largest lung cancer centers in Germany. Every year, about 600 patients are newly diagnosed with metastatic lung cancer. The physicians involved in the project are oncologists and residents in advanced oncology, working mainly at the outpatient clinic. Their daily consultations include the first disclosure of diagnosis and prognosis, disclosure of progression, and transition to best supportive care (breaking bad news). The nurses have working experience in oncology and palliative care in both inpatient and outpatient settings.
Phase 2: implementation Evaluation of communication training outcomes
Questionnaire for training evaluation To explore training, acceptance level 1 (reaction) and level 2 (learning) of the Kirkpatrick model will be evaluated [38]. The sample includes five nurses and five physicians, who will receive the training. To integrate MCA communication content, participants will complete a self-assessment questionnaire with 26 items on acquisition of intended knowledge, skills, attitude, confidence, and commitment based on participation in the training and relevance to participants' work. Items are rated on a five-point Likert scale ranging from agree completely (1), agree (2), neither agree nor disagree, disagree (4) to disagree completely (5). In addition, there are four open-ended questions on what participants liked best or least about the training, their overall impression, and what would help to enhance their communication skills. Training participants (n = 10) will be invited to complete the questionnaire after all four training sessions. The questionnaires will be analyzed using descriptive statistics, including mean, median, standard deviation, and range. Thematic analysis will be used to evaluate open-ended questions [39]. The results of each training session will be used to plan and improve the next training session.
Evaluation of implementation context and outcomes
Interviews to evaluate implementation Interviews and focus group interviews with training participants will be conducted between the training sessions to explore [40]. The main issues raised in the interviews will be recorded on a protocol sheet, summarized, and the results fed back by the researchers to the communication training and implementation team [41]. The results will be used to improve the next training sessions, to enhance implementation in everyday practice, and to adapt both MCA materials (communication manual, memory cards, questionprompt-list, and brochure) and the implementation plan following the PDCA cycle.
Quantitative content analysis of patient files From kickoff, nurses will document excerpts from the milestone conversations and follow-up calls in patient electronic files. Excerpts written 6 months after kickoff (n = 50) from the milestone conversations and (n = 50) from the follow-up calls will be analyzed for adherence to the MCA concept, shared decisionmaking, prognostic awareness, and documented topics [41]. The file content will be analyzed using a fidelity checklist of essential topics based on the MCA manual. Data will be entered into SPSS and descriptive statistics will be produced. Binary and continuous variables will be evaluated by mean, median, minimum, and maximum, and categorical variables using absolute and relative frequencies [41]. The results will help to determine to what extent the developers' intentions regarding training contents and adherence to the communication manual are seen in practice.
Phase 3: outcome evaluation
Impact of MCA on patient outcomes The effects of MCA on patients and caregivers will be determined in a RCT. Their experiences with the concept will be assessed in semi-structured interviews. The effects on interprofessional collaboration will be evaluated in a longitudinal observational study with healthcare professionals.
Effects on patients and caregivers (pragmatic RCT)
In the RCT, the effects of MCA on patients and caregivers will be assessed and evaluated with a focus on shared decision-making and on early and proactive advance care planning. Patients and their caregivers will report on daily life activities and the identification and treatment of palliative care needs. The impact of the concept regarding empathy, quality of life, and distress in patients with metastatic lung cancer will also be examined. Furthermore, needs-based use of services and the impact of further contacts in the German healthcare system will be assessed.
Primary outcome
The primary outcome is the effect of MCA on meeting patients' information needs measured using the health system and information needs dimension of the Supportive Care Needs Survey: Short Form for Patients (German version) (SCNS-SF34-G).
Secondary outcomes The secondary outcomes are the effects of MCA on perceived empathy, quality of life, and distress in patients with metastatic lung cancer and a limited prognosis, needs-based use of health services, and further contacts with healthcare professionals in the German healthcare system.
Sample and sample size The study will consist of 100 patients and 100 caregivers named by the patients. It will Data collection Recruitment of patients and caregivers will take place at the Department of Thoracic Oncology, University Hospital Heidelberg. Patients and caregivers will be approached by a study nurse who works at the department. If patients and caregivers are interested in the project, they will receive oral and written information about the study. After giving consent, baseline (t0) questionnaire data will be collected before patients are randomly assigned to a group. The first milestone conversation (tandem or standard communication with a physician) will then take place. For the follow-up, questionnaires will be distributed after 3 (t1), 6 (t2), and 12 (t3) months (Fig. 1). Study plan according to SPIRITchecklist see Fig. 2 and Additional file 2. Patient documents will be checked by a study nurse for adverse events. No adverse events are anticipated.
Randomization procedure The randomization into the groups (intervention and control) will occur in the ratio 1:1. Block randomization will be performed to ensure equal-sized groups. The randomization will be performed using sealed opaque randomization envelopes, which will be provided by the Institute of Medical Biometry and Informatics Heidelberg.
Instruments All instruments are validated in German. Table 3 provides an overview of the assessment instruments. The supportive care needs of patients will be assessed using SCNS-SF34-G. The questionnaire comprises 34 items and covers five domains: (1) health system and information, (2) psychological, (3) physical and daily living, (4) patient care and support, and (5) [42]. Subscale scores are obtained by calculating the mean of scale items. The higher the subscale score, the higher the need for support in the respective domain [43].
The supportive care needs of caregivers will be measured using the German version of the Supportive Care Needs Survey for Partners and Caregivers (SCNS-P&C-G). The multidimensional questionnaire consists of 45 items in four domains: (1) healthcare service needs, (2) psychological and emotional needs, (3) work and social needs and (4) information needs, which are assessed on a five-point scale (1 I have no problems, 2 I am already supported, 3 low need, 4 moderate need, and 5 high need). Answers 1 and 2 are grouped together so that 1 stands for "there is no need for support." For supportive care needs domains, the mean of the respective items will be calculated, ranging from 1 to 5 [44], and standardized on a 0-100 scale [45].
A patient's quality of life will be assessed using the Schedule for the Evaluation of Individual Quality of Life (SEIQoL-Q) [46], the Functional Assessment of Chronic Illness Therapy (basic module, FACT-G, and additional questions on lung diseases, FACT-L) [47,48]. The Patient Health Questionnaire (PHQ-4) [49] will be used to assess depression and anxiety in patients. The SEIQoL-Q measures each patient's quality of life by choosing, rating, and weighting five domains that patients consider important, such as family, health, or social life/other relations [50]. The five-point scale ranges from not at all (0) to extremely (100) important. Following this, patients must evaluate their overall satisfaction with the areas of life on the same scale [51].
FACT-G consists of 27 items in four domains: (1) physical well-being, (2) social well-being, (3) emotional well-being, and (4) functional well-being. All domains, except emotional well-being, consist of seven items, each with a score in the range 0-28. Emotional well-being contains six items and has a score range of 0-24. For all questions in FACT-G, a five-point rating scale from 0 to 4 (0 not at all, 1 a little bit, 2 somewhat, 3 quite a bit, and 4 very much) is used. The total score for FACT-G is calculated as the sum of the four subscale scores, provided that the overall item response is at least 80% (i.e., at least 22 of the 27 items were answered) and takes values between 0 and 108. Negatively expressed items are reverse scored prior to summing so that higher subscale and total scores indicate a better quality of life [47,48,52]. FACT-L contains the four domains of FACT-G and one lung cancer symptom-specific subscale. The seven items of the lung cancer subscale assess patient-reported symptoms, like shortness of breath and loss of weight. FACT-L is also rated on a five-point Likert-scale ranging from 0 (not at all) to 4 (very much) with a score ranging from 0 to 28 [53].
PHQ-4 is an ultra-brief self-report questionnaire that contains a two-item depression scale (PHQ-2) and a two-item anxiety scale (GAD-2) [49]. Patients assess how many times over the past 2 weeks they have felt a loss of interest and happiness, depression, melancholy or hopelessness, nervousness, anxiety or concern, and restlessness. The ordinal-scaled answer options are not at all (0), several days (1), more than half the day (2), and nearly every day (3) [54]. The total score for PHQ-4 ranges from 0 to 12, with categories of psychological distress being none (0-2), mild (3)(4)(5), moderate (6)(7)(8), and severe (9)(10)(11)(12) [55]. Patient distress will be measured using the NCCN Distress Thermometer. This assessment technique was developed by the National Comprehensive Cancer Network (NCCN) and is a brief self-reported screening instrument for recording psycho-social stress in oncological patients. It consists of a scale from 0 to 10 and a problem list. A score of 5 or higher indicates that a patient is conspicuously stressed and needs assistance [56]. If the burden is low (0-4), no additional professional support is required [56].
Data analysis All baseline patient characteristics will be analyzed descriptively. Continuous variables will be described as means with standard deviation or as medians with interquartile range, minimum, and maximum. Categorical variables will be described in absolute and relative frequencies.
The sum of the health system and information dimension score of SCNS-SF34-G is defined as the primary outcome of the RCT. The primary outcome at t1 will be analyzed using a linear model in which the outcome value is included as dependent variable and the respective baseline value and the treatment group as independent variables. Missing values in the primary outcome will be replaced by multiple imputation, which consider treatment the group and primary outcomes at t0 as independent variables using the fully conditional specification method [57]. A complete case analysis will be performed as an additional sensitivity analysis. The respective parameter estimates will be reported together with p values and 95% confidence intervals. The analysis of the primary outcomes at t2 and t3 will be done analogously to the primary linear model. The analyses of the secondary outcomes will be performed using linear (for continuous outcomes) or generalized linear models (for binary outcomes). The models include the outcome value at follow-up time t1, t2, or t3 as the dependent variable. The respective parameter estimates will be reported alongside descriptive p values and 95% confidence intervals. p values smaller than 0.05 will be considered statistically significant. Analysis will be done using the statistical software SAS v9.4 (SAS Institute, Cary, NC).
Phase 2 Implementation
Power calculation With a sample size of n = 100, a difference of 5 points on a scale from 0 to 100 for the primary outcome can be shown using a two-sided t-test at a significance level of α = 0.05 with a power of 1ß = 0.85, assuming a standard deviation of σ = 7.4 [58]. A dropout rate of 18.5% is taken into account. Prognoses of metastatic lung cancer and the experiences at the Department of Thoracic Oncology, University Hospital Heidelberg have shown that 82.5% of the patients are still alive after 3 months and 75% are still alive after 6 months. It can be assumed that the additional variance explained by the inclusion of the baseline value as a covariate will lead to increased power. The sample size calculation was performed using SAS v9.4 (SAS Institute, Cary, NC).
Effects on patients and caregivers (semi-structured interviews)
Experiences with the MCA concept regarding empathy and shared decision-making will be evaluated through interviews with patients and caregivers.
Sample and sample size Patients and caregivers who will experience MCA will be approached in the hospital setting. Patients and caregivers must be over 18 years old and able to understand and speak German well. They will be asked to participate and receive a written invitation to the study, including the background information, participation details, and informed consent form. About 12 patients and 12 caregivers will be interviewed, possibly more, until data saturation is reached.
Data collection Semi-structured interviews will be conducted at the Department of Thoracic Oncology, University Hospital Heidelberg, after at least two milestone conversations have taken place. A theory-based topic guide with open-ended questions will be used. Interviews will be digitally recorded and transcribed verbatim [59].
Data analysis Interviews will be analyzed using qualitative content analysis as described by Mayring [60]. The qualitative content analysis consists of nine steps for analyzing texts: (1) determination of the material, (2) analysis of the original situation, (3) formal characteristics of the material, (4) determination of the direction of the analysis, (5) theoretical differentiation of the question, (6) determination of the analysis techniques and definition of the concrete process model, (7) definition of the analysis units, (8) analysis steps using the category system (abstract explication and structuring) and review of the category system of theory and material, and (9) interpretation of the results in the direction of the question and application of content-analytical quality criteria. The Mayring concept is based on reducing the initial material and is, therefore, suitable for large amounts of data and systematic textual processing, as is the case in this work. Although it cannot be used for an explorative-interpretive analysis [61], the concept is well suited to answering the questions posed in this project.
Evaluation of interprofessional collaboration
Clinical employees will assess the effects of the approach on interprofessional collaboration and on the understanding of their own role within their team.
Sample and sample size All 120 team members of the medical, nursing, administration, psycho-social and therapeutic professions at the Department of Thoracic Oncology, University Hospital Heidelberg will receive oral and written information on the MCA project with the request to participate. Participants will then receive a written invitation to participate in the study, including the background information, participation details, and informed consent form.
Data collection With the invitation, participants are asked to complete an attached questionnaire prior to the first training within the MCA project (t0), directly after the implementation phase (t1), and 6 (t2) and 12 (t3) months after the implementation phase. Supportive care needs SCNS-SF34-G (for patients) [42] SCNS-P&C-G (for caregivers) [44] Patient quality of life SEIQoL-Q [46] FACT-G, FACT-L [47,48] Depression and anxiety PHQ-4 [47] Patient distress Distress thermometer [56] FACT Instrument The German version of the University of the West of England Interprofessional Questionnaire (UWE-IP-D) will be used to assess interprofessional collaboration and attitudes on teamwork of the healthcare professionals [62,63]. UWE-IP-D is a self-report instrument consisting of 34 items in a set of four scales addressing different themes. It is administered at different stages in training and education. We used three of the four scales: communication and teamwork scale, interprofessional interaction scale, and interprofessional relationships scale. Communication and teamwork items will be measured on a four-point Likert scale (1 strongly agree, 2 agree, 3 disagree, and 4 strongly disagree) leading to sum scores between 9 and 36, with scores 9-20, 21-25, and 26-36, respectively indicating a positive, neutral, or negative self-assessment of communication and teamwork skills. Interprofessional interaction and interprofessional relationship items are assessed on a five-point Likert scale (1 strongly agree, 2 agree, 3 undecided, 4 disagree, and 5 strongly disagree). The interprofessional interaction scale takes sum scores between 9 and 45, with scores 9-22, 23-31, and 32-45, respectively, indicating positive, neutral, and negative perceptions of interprofessional interaction. Sum scores on the interprofessional relationships scale vary between 8 and 40, with scores 8-20, 21-27, and 28-40, respectively, indicating positive, neutral, and negative attitudes towards the respondent's own interprofessional relationships. Additionally, healthcare professionals will report gender and profession (nursing, medical, psycho-social, therapeutic, administrative, or other allied healthcare profession).
Data analysis All the characteristics of the healthcare professionals will be analyzed descriptively. Categorical variables are given as absolute and relative frequencies. UWE-IP-D sum scores will be described as means with standard deviation and as median with interquartile range, minimum, and maximum. Differences between assessments will be analyzed using repeated-measures analysis of variance (ANOVA).
Data integration
A framework analysis will be used to integrate the data from the quantitative and qualitative research collected from different teams of researchers [64]. The following steps are taken: familiarization with the material, identifying a thematic framework, indexing, charting, mapping, and interpretation [64,65]. Emerging themes will be related to a priori identified domains [32,64,65]. Findings from all the phases will be merged using an integrative analysis [35].
Ethical aspects
Written informed consent will be obtained from each participant. Ethical approval has been given by the Ethics Committee of the University Hospital Heidelberg (S-561/ 2017). Participants can withdraw their consent at any time. Only investigators will have access to the final trial dataset. There are no contractual agreements that limit such access. Personal information and the confidentiality, coding, security, and storage of the data are in line with German privacy protection law (Bundesdatenschutzgesetz or BDSG) and the privacy policy of University Hospital Heidelberg.
Discussion
In routine practice, the care of and communication with patients with a limited prognosis is still characterized by discontinuity and lack of coordination. Inadequate communication makes coping with the realities and choices of a complex incurable disease more difficult. A longitudinal communication approach with a focus on the disease trajectory that comprises specific milestones can serve to integrate early palliative care into routine practice and can facilitate care that is individualized to patients' needs and preferences. While there are international guidelines for advanced cancer care [3,7], comprehensive implementation strategies are still lacking. As many structural and organizational aspects of national healthcare systems differ substantially, any transfer of guidelines to the German healthcare system should happen according to the specific situation and needs in Germany.
The MCA project includes the conceptualization of a communication strategy that focuses on a process in which patients and their caregivers are equally involved. The improved communication support should foster prognostic awareness and therefore, facilitate advance care planning and end-of-life decision-making.
Expected impact
The structured integrated tandem approach (physician and nurse) with interprofessional training and coaching is innovative. Consequently, the strengthening of interprofessional collaborations can be expected. The stepwise approach of the MCA project supports the communication skills and strategies of interprofessional healthcare teams involved in the care of patients with a limited prognosis. It will also enhance patients' quality of life and improve the continuity and coordination of care. The mixed-methods design will provide a detailed insight into this context, including the perspectives of professionals, patients, and their caregivers.
Limitations and strengths
A strength of this study is that it simultaneously embeds implementation and evaluation, using a strong design (randomized trial) to assess outcomes. The mixedmethods process evaluation will help in gaining a comprehensive understanding of what works and why. The weaknesses of the quantitative methods are balanced by the qualitative methods and vice versa. This exploration of a complex intervention is characterized by multiple methods and multiple stakeholders in two phases. This complexity requires a mix of researchers from different research fields with experience in qualitative and quantitative research. The phases of the MCA project build upon each other. Determining the degree to which MCA is implemented in a real-world setting will give a better understanding of the measured effects. Adaption of the interventions throughout the implementation will ensure the practicability and transferability into everyday practice.
The sample sizes of the different investigations are small but sufficient for an initial exploration of MCA in a real-world setting. However, for a definitive assessment of the effectiveness of MCA, a larger multicenter RCT is necessary. Since the same physicians will be treating patients in the intervention arm as well as the control arm, there is a potential for bias and cross-contamination between the trial arms. However, the nurses participating in the milestone conversations and offering follow-up for patients and caregivers are exclusively in the intervention arm. Our primary end point is a subjective (non-blind) patient-reported outcome, so there is a potential for bias since patients know they are in a trial and they know the purpose of the trial.
Implementing MCA and identifying relevant determinants will be explored using the example of metastatic lung cancer patients. Positive effects will be used to implement an applicable version of the concept to other medical facilities and patients with a limited prognosis.
Trial status
The beginning of recruitment for phase 2 and phase 3 is planned for May 2018, and will be completed approximately by November 2019. | 2018-08-15T15:48:42.206Z | 2018-08-14T00:00:00.000 | {
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54827288 | pes2o/s2orc | v3-fos-license | Self-completeness and spontaneous dimensional reduction
A viable quantum theory of gravity is one of the biggest challenges facing physicists. We discuss the confluence of two highly expected features which might be instrumental in the quest of a finite and renormalizable quantum gravity -- spontaneous dimensional reduction and self-completeness. The former suggests the spacetime background at the Planck scale may be effectively two-dimensional, while the latter implies a condition of maximal compression of matter by the formation of an event horizon for Planckian scattering. We generalize such a result to an arbitrary number of dimensions, and show that gravity in higher than four dimensions remains self-complete, but in lower dimensions it is not. In such a way we established an"exclusive disjunction"or"exclusive or"(XOR) between the occurrence of self-completeness and dimensional reduction, with the goal of actually reducing the unknowns for the scenario of the physics at the Planck scale. Potential phenomenological implications of this result are considered by studying the case of a two-dimensional dilaton gravity model resulting from dimensional reduction of Einstein gravity.
Introduction
Unlike quantum descriptions of the other fundamental forces, gravitation is ill-behaved.Most notably it is non-renormalizable, and as such no similar quantum description of gravity in four dimensions has been found.String theory -which is ultimately claimed to be the unifying theory of all fundamental interactions at the quantum level -requires additional spatial dimensions, that, to date, have not been verified experimentally.Alternatively the short distance behavior of the gravitational field might be improved by a mechanism called "spontaneous dimensional reduction", which opens the route towards an effective two-dimensional renormalizable formulation of quantum gravity [1,2,3,4].A complementary character of gravity at the Planck scale is "self-completeness", namely the emergence of a natural cut-off that masks spacetime pathologies in the ultraviolet regime.While each of them alone might prove to be instrumental in the formulation of a consistent quantum theory of gravity, for the first time we explore the scenario emerging from the simultaneous presence of both effects by critically reviewing currents beliefs on the subject.
Self-completeness
In the standard picture of quantum field theory, shorter length scales of a physical system become visible as one increases the energy of the probe.The Compton wavelength -representing the best possible resolution of the position of a particle -is governed by the well-known relation where M is the mass associated to the particle under consideration.The current LHC working energy ∼ 8TeV thus corresponds to matter compressed within the exceedingly minuscule distance of ∼ 10 −19 m [5].In principle one may be tempted to conclude that λ C can be arbitrarily small, provided enough energy can be supplied to a particle.By probing shorter and shorter distances, however, one enters a regime in which the background spacetime manifold becomes significantly disturbed by the energy involved in the process.Such a disturbance prevents the localization to better accuracies than a fundamental (minimal) length scale ℓ.
In other words, gravity prevents the compression of matter beyond certain distances due to the formation of a black hole.When the particle collapses to form a black hole and cannot be made smaller.Here r g = 2GM/c 2 is the gravitational radius associated to the particle mass M .Unlike in the case of the Compton wavelength, a further increase of the energy of the system will result in a bigger gravitational radius.As a result the above condition sets the minimum attainable size when both gravity and quantum mechanics are concerned.Not surprisingly, one finds and correspondingly The above relations show that gravity is self-complete, namely it protects the ultraviolet regime by setting quantum mechanical limits to length and energy.This result is often employed as an elegant argument to downplay the problem of spacetime curvature singularities, which would always be inaccessible to external probes due to the presence of an event horizon [6].This viewpoint has been reinforced by a general result obtained in an independent way by several quantum gravity modifications of black hole metrics [7,8,9]: even at the terminal stage of the evaporation the probe of the shortest scales in the vicinity of the curvature singularities would not be possible for the formation of zero temperature black hole remnants, i.e., extremal configurations occurring also in the case of non-rotating, neutral black holes [10].
We now demonstrate the robustness of this line of reasoning by its extension to the case of additional spatial dimensions, a usual requirement when considering possible quantum gravity phenomenology at the TeV energy scale.In a (d + 1)-dimensional spacetime one finds Here G d , the d-dimensional gravitational constant, reads where M d ∼ 1TeV/c 2 is the d-dimensional Planck mass and K d is a constant that varies according to the definitions of M d .The only requirement for K d is a matching between G 3 and Newton's constant G, i.e., K 3 = 1 for M 3 = M Pl. .The other factors in (7) come for the Gauss law in d dimensions.For the present discussion and without loss of generality we can simply set K d = 1 for all d as in [11].As a result the limits for energy and length turn out to be ℓ ≡ r g,min = 8 and By varying d = 4 − 10 we find ℓ = (1.23 − 2.00) /M d c and M BH,min = (π − 5.13)M d , indicating that microscopic black holes are at the reach of the typical LHC energies (see [12] for the latest production rate estimates).
Spontaneuous dimensional reduction
In the above derivation we did not invoke any specific quantum gravity character other than the fact that the energy involved are of the order of the Planck scale.The issue arises since the spacetime at this length scale might be radically different than its conventional picture.Due to its intrinsic graininess, the quantum spacetime has often been described in terms of a fractal surface which smoothly approaches the structure of differential manifold only in the infrared limit.As a result, the very concept of spacetime dimension becomes ill defined or at the very least needs revising.A reliable indicator of the dimension of a fractal is given by the spectral dimension, i.e., the actual dimension perceived by a random walker.In several approaches to quantum gravity the spectral dimension has a general form like where the infrared dimension is D IR = d IR + 1, σ is the diffusion time with the dimensions of a length squared, a and b are dimensionless constant depending on the specific model of quantum manifold under consideration [1,2,8].While at large diffusion times, i.e., σ/ℓ ≫ a, b, the spectral dimension D = D IR as expected, in the opposite regime, i.e., σ/ℓ ≪ a, b, the spectral dimension decreases to the ultraviolet value D UV = D IR − a/b.Such a behavior is connected to the potential power counting renormalizable character of gravity which in two dimensions exhibits a dimensionless coupling constant, as is evident from ( 7) for d = 1.
The desired reduction to a two-dimensional spacetime at the Plank scale fixes the range of the above parameters, i.e., a = (D IR − 2)(b + 1).Another condition can be derived from the requirement D ≥ 0: even if negative spectral dimensions are admissible for fractals, for most models of quantum gravity in the literature [1] the spectral dimension turns out to be strictly positive.As a result, one obtains that b ≥ 1 2 D IR − 1. Figure 1 demonstrates an example of dimensional reduction compatible with the above constraints (for details about its derivation in the framework of noncommutative geometry see [2]).In summary, if the quantum spacetime underwent a spontaneous dimensional reduction to D UV = 2 (d UV = 1), the non-renormalizability of gravity would just be an "apparent" low energy, classical effect [13].
Given these ingredients, the usual argument for gravity self-completeness has to be reviewed.Eq. ( 5) and ( 6) are singular for d = 1.We therefore have to reconsider the behavior of r g in lower dimensions.Before doing this we see that the gravitational potential due to the integration of the Gauss law in (1 + 1)-dimensions reads where x is the spatial coordinate and φ 0 is an integration constant.The above relation shows that the quantity G 1 M/c 2 no longer has the dimension of a length, but rather inverse length.This implies that the gravitational radius r g,1 in (1+1)-dimensions will have a intriguing new behavior, i.e.
The above result implies that in case of spontaneous dimensional reduction at the Planck scale the conventional arguments in support of gravity self-completeness is no longer valid.Such a conclusion holds for any the profile of the metric coefficients in (1 + 1)-dimensions, because the gravitational coupling G 1 becomes dimensionless and actually there is no Planck scale to discriminate between classical and quantum black holes.In addition, this fact allows one to safely employ a semiclassical model of dimensionally reduced spacetime to illustrate the associated phenomenology.
Dilaton gravity
A more detailed argument for this case may be made by considering a specific (relativistic) model of (1+1)-dimensional gravity able to circumvent the triviality of Einstein equations.This is usually achieved by the so called dilaton gravity (DG) models in which the dilaton, an extra field dated back to Kaluza-Klein theories and reappeared in the context of string theory, accounts for some key features of the higher dimensional theory (for a review see [14]).A generic action for (1 + 1)dimensional dilaton gravity is where ψ is the dilaton field and the functions U (ψ), V (ψ) are model-dependent potentials (see [15] for a comprehensive tabular summary).For the purposes of the present discussion, however, we invoke the mechanism of spontaneous dimensional reduction, d IR → d UV = 1, to determine the profile of the above potentials even if, as already stated, the choice will not restrict the broadness of our conclusions.Starting from the (d IR + 1)-dimensional action for general relativity where is the matter Lagrangian, one can show that, by expanding ( 14) in powers of (d IR − 1), the theory reduces to in the limit 16].A more general mechanism of dimensional reduction from Einstein gravity to an effective (1 + 1)-Liouville gravity have been studied, with the addition of extra terms in the action [17] where C is a constant.This enhanced model contains (15) as a subset and provides a robust match for dimensionally-reduced spherical gravity.
The above theories provide a faithful description of gravity in (1 + 1)-dimensions: they preserve classical and semiclassical properties of higher dimensional gravity [18] and can be simply connected to other dimensional reduced gravity proposals [19].By solving the corresponding field equations one finds the metric where x is the spatial coordinate and the constant C, depending on its sign, correlates to specific physical objects in our results.The horizon is thus which occurs only for C > 0. In this case, the usual self-completeness "condition" results in the mass-independent expression fixing the constant C, This is a striking result, in that it demonstrates the self-incompleteness of gravitation in a twodimensional spacetime (see right plot in Fig. 2).In the (1 + 1)-dimensional regime, the algebraic form of the defining classical quantity (the horizon radius) is equivalent to the defining quantum characteristic (the Compton wavelength).This suggests that there is no classical-quantum boundary in two-dimensional spacetime, and hence gravitation is naturally quantum mechanical at this scale.
The value and sign of C have the following repercussions.First, for C = 8π 2 one has "classical black holes", i.e., black holes that do not meet the condition for being elementary particles.Second, in a hypothetical "trans-Planckian" collision (see note 1 for the use of quotation marks of "(sub/trans)-Planckian" in such a context), the particle Compton wave length can be made smaller than /M d IR c, the d IR -dimensional Planck length, resulting in the formation of tiny, nonclassical, "trans-Planckian" black holes (i.e.M BH > M d IR ) for C = 8π 2 .Third, when C < 0, the horizon is undefined and the object is an elementary particle.
Lastly, we emphasize there is no minimum energy scale that defines a black hole.This is reflected by the fact that for a given mass M BH , it is always possible to have black hole smaller (for 0 < C < 8π 2 ) or larger (for C > 8π 2 ) of the corresponding Compton wavelength of a particle of the same mass.Note this is also true in the case of the extension of the present theory to the case of a (1 + 1)-dimensional, singularity free, non-commutative geometry: no extremal black hole configurations occur [20].Unlike in the four-dimensional (or higher) case, "sub-Planckian" black holes (i.e.M BH < M d IR ) are possible in (1 + 1)-dimensional spacetime.They cannot, however, be the direct product of a dimensional reduction mechanism (which requires Planckian energies), but rather a transient state between the formation of "Planckian" (or "trans-Planckian") (1 + 1)-black holes and their complete evaporation or some other stable configuration.
Figure 2: The scale-size in the infrared regime (left) as a function of the particle mass M , including the Compton wavelength λC (solid thick hyperbola) and the gravitational radius rg (solid linear) for classical, trans-Planckian black holes.The intersection of the above curves gives the minimum mass for forming a black hole as well as the minimum detectable length scale as it appears in the infrared limit.The direction of the arrow shows the increase of mass from sub-Planckian to Planckian scales.(Right) Scale-size in the ultraviolet regime as a function of M , where the effective spacetime dimension is 2 and gravitational radii are hyperbolae (thin solid curves).The thick solid curve represents a quantum black hole, i.e., a black hole which is at the same time an elementary particle.Classical as well quantum black holes can occur for any mass.By Hawking emission "trans-Planckian" black holes decay into "sub-Planckian" black holes according to the direction of the arrow.In both plots we adopted the units ℓ = 1, MBH,min We can see this by studying the thermal properties of these dimensionally reduced black holes.From the periodicity of the Euclidean time of ( 17) one finds the temperature which becomes T = M BH c 2 /k B by using the definition of G 1 .We see that heavier ("trans-Planckian") holes are smaller and hotter.However the positive sign of the heat capacity C ≡ ∂(M c 2 )/∂T = k B indicates that the evaporation slows down as the black hole loses energy, the contrary of what happens in the four (and higher) dimensional case.This fact results in a peculiar form of the Stefan-Boltzmann law, which in (1 + 1)-dimension reads [4] From the above relation we see that the relative mass loss 1 M BH | ∂M BH ∂t | ∼ M BH increases with the mass.Lighter black holes are bigger, colder and tend to evaporate at a slower rate.We have to restrict our analysis to mass parameters compatible with the case under consideration, however, i.e., initial "Planckian" (or "trans-Planckian") black holes with mass M BH,TP M d IR decaying into smaller "sub-Planckian" holes with mass M BH,SP < M d IR .Consequently, we can estimate evaporation times t ev for such a decay by integrating (21) within the corresponding mass interval.As a result we have that which, for M BH,SP M d IR , sets the typical time scale associated with Smaller holes would have t ev > t IR , resulting into quasi-stable objects.
To better understand the fate of such "sub-Planckian" black holes with mass M BH,SP M d IR we should go back to the mechanisms of dimensional reduction, i.e. the phase in which (trans-) "Planckian" black holes forms.The process is again regulated by the time scale t IR .The effective spacetime dimension smoothly decreases from D IR to 2 in a time lapse of the order ∼ t IR during which the system undergoes a temporary (2 + 1)-dimensional regime.This corresponds to having Newton's potential of the form where we have used the relation G 2 = 2(c 3 / )( /M Pl. c) and φ 0 is another integration constant (distinct from that in (11)).From (23) we see that the (2 + 1)-dimensional spacetime is a special case in which G 2 M/c 2 is a dimensionless quantity, signaling the absence of event horizons.This fact is in agreement with black holes derived from the BTZ action, that can only exist in anti-deSitter universes [21].In addition Newton's potential is divergent both in the UV and IR regimes, a borderline situation between IR-dimensional and UV-dimensional cases.As a consequence we interpret the (2 + 1)-dimensional case as the regime of a phase transition between the higherdimensional and the two-dimensional black holes.
In light of this reasoning we can argue that "sub-Planckian" black holes would not completely evaporate, but they would rather undergo to a "dimensional oxidation", namely the opposite process to the dimensional reduction.In other words the initial (trans-) "Planckian" black holes would decay into transient "sub-Planckian" black holes in a time t ev ∼ t IR to oxidate into ordinary sub-Planckian particles in the (d IR + 1)-dimensional spacetime.This might change the conventional signatures for black hole detection in particle accelerators [22] and explain the reason why latest data tend to exclude their observation [23].
Conclusions
In summary, we have presented in this paper new insights on the nature of lower-dimensional gravitation, specifically that the well-known self-completeness condition does not hold in spacetimes below (3 + 1)-dimensions.The immediate consequence of this result is that the mass of a quantum black holes formed in a (1 + 1)-dimensional Universe is unbounded from below.Consequently, this would suggest that evaporating black holes will eventually reach a new non-thermal phase in their evolution if Planckian dimensional reduction theories are correct.For primordial black holes of the appropriate masses, this has already occurred.Such objects can thus be considered a new catalyst in early Universe physics such as formation mechanism, a possible new candidate for dark matter, or even a new bi-product of high-energy collisions at the specified energy.Depending on the scale of such transitions, physical evidence could be just within reach of present or future collider experiments, ultra-high energy cosmic ray detectors, of other cosmological probes.If detected, signals of dimensional reduction would present a profound confirmation of these innovative formulations of spacetime structure, and would revolutionize our understanding of the Universe.
Although the above scenario is intriguing, we do not wish to further speculate without a corroboration from a more detailed analysis.In this paper, we safely prefer not to assume an a priori position but rather we simply conclude that self-completeness and dimensional reduction are conflicting occurrences, connected by an "exclusive disjunction" or "exclusive or" (XOR).This logic is instrumental for reducing the variety of possible approaches in the description of physics at the Planck scale.
Figure 1 :
Figure 1: Example of dimensional reduction [2]: the spectral dimension D as a function of the diffusion time σ for DIR = 4.A decrease of spacetime dimension already occurs in the sub-Planckian regime, i.e., σ > ℓ 2 , in order to match the value D = 2 for σ = ℓ 2 .As a consequence semiclassical arguments are still valid for 2 < D < DIR. | 2013-06-29T11:26:52.000Z | 2012-06-20T00:00:00.000 | {
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231598242 | pes2o/s2orc | v3-fos-license | Affecting Argumentative Action: The Temporality of Decisive Emotion
This paper explores the interrelations between temporality and emotion in rhetorical argumentation. It argues that in situations of uncertainty argumentation affects action via appeals that invoke emotion and thereby translate the distant past and future into the situated present. Using practical inferences, a threefold model for the interrelation of emotion and time in argumentation outlines how argumentative action depends on whether speakers provide reasons for the exigence that makes a decision necessary, the contingency of the decision, and the confidence required to act. Experiences and choices from the past influence the emotions experienced in the present and inform two intertemporal mechanisms that allow speakers and audiences to take the leap of faith that defines decision-making under uncertainty: retrospective forecasting and prospective remembering. Retrospective forecasting establishes a past–future–present link, whereas prospective remembering establishes a future-past-present link, and, together, the two mechanisms provide a situated presence that transcends the temporal constraints of uncertainty. Finally, the applicability of the model is illustrated through an analysis of a speech delivered by Danish Prime Minister Mette Frederiksen at the outset of the COVID-19 pandemic, a time where the need for decisive, yet argumentative action was crucial.
1 3 minister and leader of the Social Democratic Party, Mette Frederiksen, thus began what politicians, industry leaders, and commentators shortly after dubbed "a historical press conference" (Schulz 2020), stating: "What I will say tonight is going to have major consequences for all Danes." She then went on to announce the most drastic lockdown of Danish society in peacetime.
Forty-four minutes later, an opposition member of the Danish Parliament, Mette Abildgaard of the Conservative People's Party, tweeted: "Good press conference by the Prime Minister. Will possibly hate myself for this tweet at the next election, but I trust her as prime minister in these very serious times." 1 Abildgaard's tweet illustrates that while emotions may exist and change across time (present trust, future hate), they also shape opinion and agency in the present. To make decisions under uncertainty is to feel one's motives well up inside oneself and then act upon them (Helm 2009). While the safest bet for any decision maker might be to hold out for more data and their tantalizing promise of predictability, novel and uncertain situations amplify the dilemma between an epistemic waiting game and a prudential willingness to act incisively. Existing argumentation research suggests deliberation about choice of action (Kock 2017) under uncertain circumstances (Walton 1990;Tindale 2018) define rhetorical situations and rhetorical argumentation, essentially, as "The Realm of the Uncertain" (Kock 2020, p. 288).
Uncertainty has a both epistemic and practical character in rhetorical argumentation (Zarefsky 2020), which emphasizes the critical importance of time because a practical choice has prospective outcomes, whereas demonstration leads to true conclusions, independent of the passing of time (Perelman and Olbrechts-Tyteca 2020). Indeed, emotions are inevitable, especially in situations of uncertainty, but a person's decision-making capacity also depends on them (Damasio 1994). Building on Damasio's groundbreaking work, Barrett underlined: "Affect is not just necessary for wisdom; it's also irrevocably woven into the fabric of every decision" (2017, p. 80). 2 The rhetorical tradition has always embraced emotion in persuasion (Katula 2003), just as it recognizes the centrality of time to persuasion (Miller 1994;Tindale 2018, p. 182). Although Perelman and Olbrechts-Tyteca hinted at the emotional nature of temporality in argumentation (2020, p. 319), and emotion scholars mentioned the past and future orientations of emotion (e.g., Helm 2009;Lerner et al. 2015), the temporality of appeals to emotion in argumentation studies remains 1 Unless otherwise stated, I have translated all quotes. Where necessary, I explain the reason for using a specific word. In this case, Abildgaard used the Danish word "tryg," which in this context translates as "trust". "Tryg" stems from Old Norse, "tryggr," and German "true," underlining the etymological connection with trust (Den Danske Ordbog 2020). One could also translate "tryg" as confident, because confidence stems from the Latin confīdere, that is "to put trust in, have confidence in, be sure." (Merriam-Webster 2020). 2 In line with a well-established distinction within emotion research, I rely on affect as an umbrella term covering mood and emotion, in which emotions are discrete and intense but short-lived experiences, and moods are longer, more diffuse experiences that lack an awareness of the eliciting stimulus (Elfenbein 2007).
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Affecting Argumentative Action: The Temporality of Decisive… largely unexplored. 3 Scott has recently encouraged further research "thematizing the essentially temporal idea of ethos" (2020, p. 35), but he and other argumentation scholars appear silent about the need to connect pathos and temporality in relation to decision making. This paper seeks to shed light on this blind spot by exploring the connection between emotions and time in argumentation.
This aspiration begins with Micheli's call to further examine "the discursive constructs of situations and their emotional orientation" (2020, p. 15). Such discursive constructs of situations involve not only the present situation but also future projections, which argumentation may affect and act as grounds for choosing one option over another. Given that decisions happen in the now, one must understand how speakers successfully make the future--which their decisions will affect--present, using the past as a central resource. My main argument in this paper is as follows: In uncertain situations, argumentation affects action via appeals that invoke emotion and thereby translate the distant past and future into a situated present. Emotions make arguments about the future appear present, creating an opportunity for action that enables people to believe in and act on them.
I seek to contribute to rhetorical argumentation in two respects. Theoretically, understanding the temporality of emotion can strengthen our appreciation of the logos of the passions (Brinton 1988a;Waddell 1990;Micheli 2010), which, I argue, is necessary in any deliberation about choice where emotions and incommensurable values render a common yardstick for reaching a "true" conclusion futile (Kock 2017, p. 60). Societally, the year 2020 marks the outbreak of a global pandemic and the rise of a social movement against systemic racism, not to mention an ongoing climate crisis. Such consequential global crises stir the emotions, and emotions must be harnessed rhetorically to engage citizens in both the necessary decision making and to mobilize support for solutions. Now more than ever, it is apparent that emotions inevitably influence decision making (Vohs et al. 2007); the question is how to harness them rhetorically in a way that enables such decision making to be wise.
In terms of making a conceptual contribution, a three-fold model for interrelating emotion and time in argumentation can illustrate how speakers must provide reasons for (i) the exigence that makes a decision necessary, (ii) the contingency of the decision, and (iii) the confidence required to act. Experiences and choices from the past influence the emotions experienced in the present and inform two intertemporal mechanisms that allow speakers and audiences to take the leap of faith in decision making: retrospective forecasting, which establishes a past-future-present link, and prospective remembering, which establishes a future-past-present link.
To investigate the connection between temporality and emotion in argumentation, I first review the roles of time and emotion in argumentation, and then combine insights from the two strands of argumentation theory to substantiate my synthesis 3 In a recent special issue of Argumentation on time and place (Tindale 2020), emotions play an insignificant role despite their role in practical argumentation that focuses on the future (e.g. Walton 1992Walton , 1996Tindale 2018, chapter 8;Kock 2017). However, see Cigada (2006) for a valuable exception as well as Macagno and Walton (2014, p. 68) for a brief mention in addition to Walton's work on emotional appeals in relation to traditional fallacies (1997,2013). and propose a conceptual model of temporality and emotion in rhetorical argumentation. To illustrate the empirical import of the theoretical work, I have focused on the COVID-19 pandemic, for this sudden and dramatic development has already profoundly affected societies, putting humanity on an impending "tightrope walk to recovery" (OECD 2020). As such, the coronavirus crisis also provides a pertinent lens through which to understand how people interact and reason about which decisions to make and how to act in a situation marked by high uncertainty. To illustrate this applicability, I briefly analyze Danish Prime Minister Mette Frederiksen's opening speech at the March 11 press conference.
Temporality and Emotion in Argumentation
Argumentation is an unfolding process in which the audience is an active participant, not a "mere passive receptor" (Tindale 2018, p. 30). Although I emphasize this aspect of audience agency because of its prevalence in contemporary rhetorical theory (Hoff-Clausen 2018), I also stress that creating adherence in decision making contexts depends on whether people are committed to carrying out the (future) actions they decide on in the very present (Scott 2020). The uncertain nature of rhetoric makes time an essential factor (Zarefsky 2020, p. 301). Humans do not deliberate about matters where their words have no power, but a rhetorical situation (Bitzer 1968) implies an exigence, an urgency-laden imperfection that the audience, here defined as a mediator of change, possesses the agency to resolve, despite the existence of various constraints that reflect uncertainty about the outcome of the decision. Largely because of this uncertainty, emotions play an important role, as they emphasize salient agentic clues about what to do (Pfau 2007). The following sections briefly present contemporary conversations on temporality and emotion in argumentation to provide a foundation for developing the subsequent synthesis.
Temporality in Argumentation
Time and temporality are not synonymous. Rather, temporality is the "negotiated organizing of time" (Granqvist andGustafsson 2016, p. 1009) that establishes "ongoing relationships between past, present, and future" (Schultz and Hernes 2013, p. 1). This definition stems from organization studies but clearly resembles that used in Perelman and Olbrechts-Tyteca's seminal paper on rhetorical argumentation, in which temporality is "the intervention of time": The oppositions that we notice between classical demonstration, formal logic, and argumentation may, it seems, come back to an essential difference: time does not play any role in demonstration. Time is, however, essential in argumentation, so much so that we may wonder if it is not precisely the interven-
3
Affecting Argumentative Action: The Temporality of Decisive… tion of time that best allows us to distinguish argumentation from demonstration. (2020, p. 310). 4 I emphasize that the "intervention of time" plays an essential role in distinguishing argumentation from demonstration and stress that rhetorical argumentation revolves around practical choice (Kock 2017). Furthermore, where demonstration leads to true conclusions, independent of the passing of time, argumentation is an action one performs with words when seeking adherence to a proposal. Seeking adherence concerns influencing an audience to make a decision that will impact the shape of an unknown future. Hence, the notion of "argumentative action" (Perelman and Olbrechts-Tyteca 2020, p. 316) underlines the dynamism of persuasive symbolic action, which provides compelling reasons both for taking action and for the very action that stems from such argumentation.
A key aspect here is the question of how the concept of temporality, as a constituent part of argumentation, is capable of "translating" or moving the past and future into the present: "Argumentation confers simultaneity on elements that normally would be distant in time, a simultaneity that derives from their integration in a system of ends and means, of projects and obstacles." (Perelman and Olbrechts-Tyteca 2020, p. 329).
This simultaneity exists when an audience comes to understand that the decisions it makes have future consequences, vague though such distant futures might seem when viewed from the present: the future simply lacks presence, one could say. The ability to invoke presence, a key term in Perelman and Olbrechts-Tyteca's theory of rhetorical argumentation (1969, p. 115), is crucial in argumentation involving future considerations. Persuasion hinges on the question of how imagination of the future becomes present in the moment of deliberation. As a rhetorical ability, then, creating presence revolves around the choice of certain salient elements and their presentation to the audience, as persuasive appeal arises from the importance with which a speaker endows these elements simply by choosing to focus on them (Perelman and Olbrechts-Tyteca 1969, p. 116).
When a speaker focuses on certain elements, creating a salience in the presence anticipating what is yet to come and how choices can impact such a foreseen future, the concept of prolepsis is worth mentioning, as it "allows our attention to be directed to particular deliberative ends" (Mehlenbacher 2017, p. 246). Stemming from the Greek word prolambanein, to anticipate, (Walton 2008, p. 144), proleptic argumentation can be understood as both a rhetorical figure anticipating a premise yet-to-happen and a subsequent consequence (e.g. 'If you tell mom, I will never help you again ') and several argument tactics distinguished by their varying certainty of future outcomes. Prolepsis can namely be both (i) an anticipation and rebuttal of an opponent's argument, (ii) a certain prediction of future events, and as iii) presage, a forewarning of a potential future (Mehlenbacher 2017, p. 235); the latter being highly relevant to the current paper, and an aspect, which I shall return to in Sect. 3.1.
To summarize, although people exchange arguments in the ongoing present, rhetorical argumentation aims at the future, yet draws on the past. Given the foundational role of emotions in decision making (Damasio 1994;Barrett 2017), we ought to also ask how emotion and argumentation are related.
Emotion in Argumentation
When time is limited and outcomes are contingent on decisions, emotions affect decision making (Pfau 2007), but such decision making is therefore not irrational. A key assumption is that reasonable grounds for an emotion can exist, so emotion can hence function as a legitimate reason for action (Greenspan 2004;Nussbaum 2015).
Emotions are "adaptive responses to the demands of the environment" (Elfenbein 2007, p. 316), and since antiquity such responses have figured in reasoning about actions because "emotions are all those feelings that so change men as to affect their judgements, and are accompanied by pleasure and pain" (Aristotle 2005, 1378a20). Speakers may argumentatively describe and construe such environmental demands as establishing a connection between the situation, the audience's values, and the need to react to those values. To assess a situation as "good" or "bad" and hence worth approaching (pleasure) or avoiding (pain), an audience must have a system of values that provide reasons to desire and act in ways that achieve the goals or avoid the threats corresponding with those values (Macagno and Walton 2014, p. 65).
The inclusion of emotion in decision making is a source of long-standing dispute between rhetoric and ethics, because emotions can indeed prompt one to act with affect without considering the ramifications. The challenge is to distinguish wellgrounded emotional appeals from manipulative trickery. As Villadsen aptly noted: Persuasion may as well be used to inflame passions and cloud judgment as it may speak to reason and justice. With rhetoric there is always the threat of deterioration into deception and manipulation, but it is accompanied with the possibility of insisting on sound reasoning and relevant emotional and moral appeals. (Villadsen 2016, p. 48).
As emotions and values are necessary and unavoidable in rhetorical argumentation about practical choice, below I describe how the rhetorical tradition has conceptualized appeals to emotion (pathos).
Although Aristotle underlined that the speaker should put "the audience into a certain frame of mind" (2005, 1356a2), several scholars (Lee 1939;Brinton 1988b;Micheli 2010;Welzel and Tindale 2012) have pointed out his telling vagueness on exactly how a speaker stirs an audience's emotions. However, as Brinton explained: "Generally by pathe Aristotle means (in the Rhetoric at least) feelings which influence human judgment or decision making and which are accompanied by pleasure or pain" (1988b, p. 208). Yet, when a speaker presents an argument capable of stirring, say, confidence within an audience (confidence, according to Aristotle, being the opposite of fear), but uses factual grounds to do so, logos and pathos seem difficult to separate. Simply put, "logos and pathos interact in that emotional appeals are generally built on a rational foundation; conversely, logical appeals generally have an emotional component" (Waddell 1990, p. 383). This type of interaction echoes another ancient scholar, namely Quintilian and his advice on making facts come alive before the eyes of an audience in order for them to 'feel' their relevance to a given case (see also Katula 2003, p. 9): It is a great gift to be able to set forth the facts on which we are speaking clearly and vividly. For oratory fails of its full effect, and does not assert itself as it should, if its appeal is merely to the hearing, and if the judge merely feels that the facts on which he has to give his decision are being narrated to him, and not displayed in their living truth to the eyes of the mind. (Quintilian 1922, p. 245).
Brinton labeled such interaction of logos and pathos a pathotic argument, understood here as a "drawing of attention to reasonable grounds for the passion or emotion or sentiment in question." (Brinton 1988a, p. 79). Hence, a pathotic argument includes a dimension of reason-giving for why a certain emotion (or combination of emotions) is appropriate, and these reasons allow one to examine emotion as lending an argument acceptability, relevance, and adequacy (Gilbert 2004).
Still, emotions have several functions in argumentative contexts (Carozza 2007) and a variety of normative roles. The dominant view within argumentation and logic has seen appeals to emotion as fallacies. Take, for instance, fear appeals that impose a threat on an audience and function as an argumentum ad baculum (Walton 1996). However, as Govier (2010), O'Keefe (2012) and Walton (1992Walton ( , 2013 have all argued, appeals to emotion such as fear are not necessarily fallacious and are thus not per se unreasonable, because they "invoke consequences of an action as a basis for justifying performing or not performing that action" (O'Keefe 2012, p. 27).
According to Micheli (2010), in a "traditional" view emotions function as adjuvants to argumentation, meaning that speakers can appeal to emotions to support a conclusion and thereby promote a judgment, decision, and potentially action. In the convergence between judgment and emotion, I should underline, both are equally important. Emotions affect people's cognitive judgments, as Aristotle recognized, for "when they feel friendly to the man who comes before them for judgement, they regard him as having done little wrong, if any; when they feel hostile, they take the opposite view" (Aristotle 2005, 1378a35). However, cognition can also affect emotion, because the emotions that affect decisions arise from grounds pertaining to "the role of judgment in the formation of the passions" (Micheli 2010, p. 6).
This dynamic understanding, in which emotions have not only cognitive effects but also cognitive origins, provides an important bulwark for assessing emotions as legitimate reasons. For the present purposes, I focus on argumentation that enables an emotional experience to be rooted in the Aristotelian cognitive understanding of emotion (Morreall 1993, quoted in Pfau 2007. In relation to arguments, emotion is defined as a specific state of mind directed at others and based on the grounds on which the emotions arise and thereby lead to persuasion. 5 If the grounds for an emotion are reasonable, then such an emotion can also be a legitimate reason for judgment and action (Greenspan 2004). Because beliefs and cognition can both function as grounds for emotions and give rise to them, it can be helpful to distinguish between evoking and invoking emotion (Brinton 1988b). Evoking emotion is an appeal toward emotion, an endeavor to arouse that emotion in the audience and thus cause an action, but not per se to provide a reason for taking it, as in 'reflex emotions' defined as "fairly quick, automatic responses to events and information" (Jasper 2011, p. 287). Invoking emotion is an appeal to emotion that involves a reason on which to base an action, which is to say the speaker gives the audience a reason to feel a certain way on which it can act. In short, to invoke emotion reflects how reasoned emotion can prompt responsive action. As such, adhering to a cognitive theory of emotion enables one to view emotion as reasonable in the dual sense of its providing reasons and being grounded in reasons. Having described the roles of temporality and emotion in argumentation let me unfold my main argument.
The Temporality of Emotion in Argumentation
Argumentation affects action via appeals that invoke emotion in order to translate the distant past and the anticipated future into the situated present. Such appeals function more than simply persuasively when a speaker appeals to a specific emotion, for an argument that succeeds in invoking an emotional focus can impel an audience to commit to action because of the expected consequences vis-à-vis past experiences (Walto 2002). As such, an argument has import to those making the decisions, thus motivating them to take action (Helm 2009). For example, to invoke patience persuasively, one must illustrate-that is, provide reasons in support ofthat an impending mission is of a magnitude requiring a long, sustained effort, yet is both possible and worthwhile-and, hence, merits patience.
The temporality of appeals to emotion remains underexplored in argumentation studies. However, there are notable exceptions: Perelman and Olbrechts-Tyteca hinted at the emotional nature of temporality in argumentation when referring to "the insistent [appuyé] style, meant to provoke emotions, mainly aims to frame thought" (2020, p. 319). Macagno and Walton underlined that "emotions are both the result of past choices and past experiences, and evaluations of present and future state of affairs" (2014, p. 68), further underscoring the temporal dimension of emotions in relation to decision-making that in the case of for instance fear often involve "a choice between long-term safety and immediate gratification" (Walton 2013, p. 23). Mehlenbacher pointed to the underlying emotional nature of reasoning based on anticipation (prolepsis), in the sense that an anticipation of uncertain but imaginable 1 3 Affecting Argumentative Action: The Temporality of Decisive… outcomes "allows us to determine our current position in terms of desires, reason, and emotion for deliberation about prospective outcomes in terms of current actions or choices." (2017, p. 246). Scott (2020) explored the "internal temporality" of argumentation, understood as the temporal unfolding of the involved actions associated with argumentation, such as speaking, listening, doubting, and judging (p. 33), although he only briefly tied temporality to emotion in argumentation. In fact, the following passage is the only place in Scott's paper where he explicitly mentioned affect (neither pathos nor emotion appear in the paper): The concept of adherence is essentially temporal-in the same way that something like a promise cannot be understood without a temporal reference to a possible future where it is either honoured or broken. With respect to adherence, this is to say that what a person is intellectually and affectively committed to at a given point in time cannot be reduced to any particular "present." (Scott 2020, p. 31).
Indeed, adherence depends on both intellect and affect. Moreover, as should be evident by now, a logos of the passion and a passion of the logos converge (Waddell 1990). The notion of adherence, which Perelman and Olbrechts-Tyteca (1969, p. 1) stressed as fundamental in rhetorical argumentation, is highly relevant in a decision-making context of uncertainty. To adhere to a proposal-say, deciding to keep physical contact to a minimum-is to accept intellectually and affectively that the grounds on which the proposal rests are sufficiently convincing at the time the proposal is made, its building on existing knowledge and experience. By drawing on the past and imagining the future to inform the present in which a decision takes place, the temporality of argumentation gives presence to this moment, but how can one fully grasp such a presence without considering emotions and their temporal orientations?
The rest of this section proceeds as follows: First, a synthesis of temporality and emotion shows how temporal orientations of emotions affect rhetorical argumentation. Second, a conceptual model provides two temporal mechanisms for invoking presence. Third, a brief analysis of the speech in which Mette Frederiksen announced the Danish lockdown illustrates how the model works and may aid future theorizing of the temporality of decisive emotion.
The Temporality of Decisive Emotion
Emotions are "energy for action" (Plantin 1998;in Cigada 2006), and decisions made under uncertainty require a willingness to act on arguments despite a lack of sufficient data. As such, the temporality of argumentation touches upon the ontological duality of rhetoric (Bitzer 1968;Vatz 1973). When a speaker discursively makes the present moment appear to be the right moment in which to act, she draws on the mutual interconnectivity of the past and the future (Miller 1994). To make a decision in the present that will affect the future is to argue why the very targets to which people react with emotion warrant attention and action (Helm 2009, p. 250).
An Appeal to Emotion Appeals to Time
Before unfolding the temporal orientations of emotions, I would like to highlight why import is central to a theory of rhetorical argumentation. Something has import when it is worthy of attention and action, thus leading a person to be "reliably vigilant for circumstances affecting it favorably or adversely and be prepared to act on its behalf" (Helm 2009, p. 250). Feeling the motivational "pull" of emotions is an aspect of evaluating how to respond to surroundings that impose meaning on humans. One can therefore view appeals to emotion as appeals that invoke an emotional focus of import to decision makers and therefore resonate with the cognitive evaluations (appraisals) arising in the immanent situation and affecting the experience of emotion, which in turn motivates a person to decide and act. As Micheli wrote, such cognitive criteria of evaluation involved in experiencing emotion "offer interesting cues for the study of the discursive and emotionally-oriented constructs of events and situations [italics in the original]" (2020, p. 15). Of particular importance to a rhetorical understanding of emotion are the appraisals by which a person evaluates the environment and interaction with other persons (such as the speaker or the deliberating audience), motivational action tendencies, and the subjective experience of feelings (Moors et al. 2013, p. 119). Appraisals could encompass goal relevance (I must act to protect what I value), agency (my actions matter), certainty (amidst uncertainty, some signs give me a degree of faith), and coping potential (I have the means to withstand an enemy that initially frightened me).
These are all felt evaluations that undergird how it feels to be in a situation illuminating that the "discursive dimension of emotions appears with a particular clarity when emotion is in debate." (Plantin 1999, p. 4). In other words, to feel an emotion like anger, a person will perceive negative events as being predictable, under their own human control (agency), and brought about by others, which may lead that person to engage in riskier behavior because she perceives little risk (Lerner and Keltner 2000). Here, agency comes to the fore in terms of whether audience members feel they can actually do something about the matter at hand. From a temporal perspective, human agency is A temporally embedded process of social engagement, informed by the past (in its habitual aspect), but also oriented toward the future (as a capacity to imagine alternative possibilities) and toward the present (as a capacity to contextualize past habits and future projects within the contingencies of the moment). (Emirbayer and Mische 1998, p. 963).
Considering that rhetorical agency is defined as "the relative capacity of speech to intervene and affect change" (Hoff-Clausen 2018, p. 287), I would like to stress the link between the inherent temporality of agency and the role emotions play in rhetorical argumentation. If a speaker is to convince decision makers to decide and even act, and this commitment requires some assessment of agency, several emotions may arise and exist simultaneously. "In short, to feel one emotion is to be rationally committed to feeling a whole pattern of other emotions with a common focus" (Helm 2009, p. 251). Crucially, these patterns of emotions-arising from appraisals of the situation-stand in relation to the temporal orientation of the emotional focus, and 1 3 Affecting Argumentative Action: The Temporality of Decisive… the reasonableness of such practical emotional patterns depend exactly on their past (and expected) reason-giving capabilities: Emotions serve to "mark" practically significant thoughts with bodily (and hence affective) indicators of past experience. According to an evaluative account, characteristic thoughts have come to be contents of emotion-and part of what identifies them as the types of emotion they are: fear, anger, joy, pride, and so forth. (Greenspan 2004, p. 208).
To summarize, appeals to emotion can invoke an emotional focus of import to decision makers, and when such import resonates with cognitive evaluations of the past, present, and future, emotion becomes timely and potentially reasonable.
Temporal Orientation of Emotion in Argumentation
When one includes the passing of time and events, the multidimensionality of emotion, which rarely exists independently, becomes part of rhetorical argumentation. For instance, a well-grounded fear of COVID-19 will tend to change as time progresses and events unfold, turning into relief or joy if people avoid becoming sick, disappointment or even grief if they do not, or anger if someone (un)knowingly endangers others, thus making all physical distancing efforts seem worthless. The temporal aspect of accumulating evidence will, then, help determine whether initial fear turns out to continue to be well-grounded as new information, experience and knowledge either harness the robustness of that emotion hereby underlining the rational (cognitive) structure of emotions (Micheli 2010, p. 6) or lead the rational actor to acknowledge that she did act in good faith but with time should abandon her continued commitment if there eventually is a lack of support for an anticipated future emotion. To continue along this path of commitment, one can view an initial well-grounded fear as a rational strategy of pre-commitment, prompting action, that (should) only hold as long as there are sufficient reasons in favor of supporting continued commitment: In such cases [where wished for outcomes only materialize after a long investment period], the rational entrepreneur would not ignore sunk costs. But she would not be too highly swayed by them either, and would only base her calculations on commitment to realistic prospects of future success or failure, judged by practical reasoning. (Walton 2002, p. 499).
Emotions have temporal orientations enabling us to make a preliminary distinction between future-and past-oriented emotions in argumentation by drawing on Helm (2009), Baumgartner, Pieters, andBagozzi (2008), and Cigada (2006). Notice that the above emotions are bound together by a common focus of import to the people experiencing them. This binding allows one to view appeals to time as appeals to the interaction between past-and future-oriented emotions and how these make the present worthy of attention and action.
Helm (2009) discussed eight such emotions, distinguishing between positive and negative past and future orientations; for example, satisfaction has a positive past orientation, and fear a negative future one. In a study on emotive communication in the political aftermath of World War II, Cigada (2006) further distinguished between the near-past and distant-past positive (euphoric) and negative (dysphoric) emotions. She underlined that pride in a historic tradition of working to ensure freedom and human rights functions as a particular argument in favor of hope about a future political situation; for example, if we won our freedom in the past, we can re-win it. This perspective emphasizes the dual argumentative understanding of emotion as both providing reasons to support a conclusion and functioning as a conclusion (Micheli 2010). Emotions can draw their reasonableness from the re-presentation of shared past events--which function as cause for, say, pride--and from imagined future events, which in turn support a focus on the action proposed in the present.
However, future-oriented emotions are both anticipatory--that is, felt in the present--and anticipated, in other words, to be felt in the future (Baumgartner et al. 2008). Anticipatory emotions such as hope or fear arise in the present at the prospect of a desirable or undesirable future event, whereas anticipated emotions stem from an imagined sense of how experiencing certain emotions will feel once future events have occurred. From an argumentative perspective, both forms of emotions function to provide an affective component when the consequences of an action are rhetorically deployed as a justification for taking or not taking that action. The interplay between instilling beliefs about anticipated (future) emotions and arousing current anticipatory emotions revolves around both the prospects of the subsequent diminishment or fulfillment of those very emotions and the reasons why they arose or are expected to arise (O'Keefe 2012, p. 28).
The temporal orientations of and relation between emotions brings me to the importance of balancing competing emotions. Sheer terror, for example, can be paralyzing. Pfau (2007) provided an elegant account of how fear and courage interact in what he labels "civic fear", or fear that leads one to deliberate on, recognize, and ultimately respond to or confront contingent events that decision makers find reasons to deem worthy of fear. Similarly, Mehlenbacher's account of the practical inference linking anticipated (proleptic) future outcomes and present action underlines that the issue at stake has to be proximal, have implications to the lives of the decision makers, in addition to "uncertain but imaginable outcomes." (2017, p. 246). When those conditions are established, first, the speaker must be able to portray a dangerous target as a spatially and/or temporally proximate threat to decision makers, for if it will have no apparent impact on their well-being, no action is required. Second, and equally important, one must convey that the object of fear is contingent rather than inevitable, to ensure that decision makers believe that taking action could enable them to avert the threat that constitutes their fear. Third, the speaker must encourage decision makers to believe that they are, in fact, capable of taking worthwhile action.
In summary, emotions have temporal orientations and become interwoven as time unfolds. In other words, they do not exist independently of each other, but depend on their temporality and the appraisals with which speakers situate emotions in moments of time. For instance, a person experiencing fear in the present might soon experience the past-oriented emotion of relief if the source of fear proved not to inflict the anticipated pain (Clore and Ortony 2000).
Model: Affecting Argumentative Action
Building on the idea that emotions have temporal orientations as described above, a threefold pathotic argument outlines how a speaker must present her specific reasons for a decision in a way that convinces an audience to make that decision. The argument must therefore express (i) the exigence that a decision is necessary, (ii) the contingency of the decision, and (iii) the confidence to act. The pathotic argument enables us to present the following conceptualization of temporality and emotion in rhetorical argumentation (see Fig. 1): In the following, I explain the concepts and mechanisms of the model. Argumentatively, the model reflects two interacting practical inferences (Walton 2006, p. 300) entailing (a minimum of) two temporally linked scenarios. I build on Mehlenbacher's suggestion to distinguish between "prolepsis-with-negative-future and prolepsis-with-positive-future" (2017, p. 246), and therefore distinguish between two scenarios (broadly depicted as positive or negative, although I also acknowledge that this distinction may not hold when being exposed to empirical scrutiny and complex causal chains) that follow from either making a decision or continuing with the status-quo (in-decision). Nonetheless, for conceptual purposes, one scenario involves a future goal, G (worth achieving), which the audience can help realize if making the present proposed decision, D. The goal, G, reflects positive future-oriented emotions. The other scenario involves a goal, G', deemed worth avoiding, which maintaining the status quo--an in-decision, D'--will most likely lead to (hence, the Fig. 1 The temporality of affecting argumentative action negative emotions). In both scenarios, experiences and choices from the past influence the emotions experienced in the present (Macagno and Walton 2014, p. 68) and inform the two intertemporal mechanisms of retrospective forecasting, which establishes a past-future-present link, and prospective remembering, which establishes a future-past-present link. Although Fig. 1 only depicts retrospective forecasting as negative and prospective remembering as positive, both mechanisms can rely on positive and negative valences as well as interact; that is, (reasonable) fear of negative future goals (G') can lead to a decision (D), which eventually leads to positive future outcomes (G) exactly because of that decision. Epistemic and practical uncertainty mean that the inference linking a present decision with a future goal will never be conclusive. The inference is quasi-logical (Perelman and Olbrechts-Tyteca 1969, p. 193), and adherence depends on whether the audience accepts the temporal interval between decision and consequence, that is, "the indeterminate wedge between cause and effect" (Bolduc and Frank 2010, p. 313). Even in cases where the consequences are near-certain, or what Mehlenbacher refers to as Prolepsis as future anteriority, an argument that anticipates and establishes a future fact (2017, p. 244), incommensurable values still guide decisions (Kock 2017, p. 68). Hence, the model seeks to illustrate how a speaker might use experiences and choices and thus accumulated knowledge of the past (EC/EC') to inspire confidence in making a decision (D) in the present by invoking futures worth achieving (G) and/or avoiding (G'), all as part of the process of making those very outcomes contingent on the advocated decisions. The concepts of the rhetorical situation (Bitzer 1968;Vatz 1973) and Pfau's (2007) "civic fear" framework for providing a constructive way of urging an audience to deliberate and take action guide the following conceptualization.
Exigence or the Need to Make a Decision
First, the speaker must diagnose the current situation as one requiring a decision. In situations characterized by high uncertainty, the existing data might dictate that inertia is the only "logical" choice, as nothing in the existing circumstances warrants change (Perelman and Olbrechts-Tyteca 1969, p. 106). Yet, the speaker is convinced that action and thus a deviation from the known path are required. In rhetorical terms such a need to act presents an exigence defined as an "imperfection marked by urgency; it is a defect, an obstacle, something waiting to be done, a thing which is other than it should be" (Bitzer 1968, p. 6). However, both situation and discourse may constitute such urgency (Vatz 1973;Leff and Utley 2004), especially if a speaker encourages present decisions whose consequences remain to be seen--economic reform policies, for example. Therefore, the question remains; how does a speaker "prove" a specific action is necessary, let alone argue in favor of taking it, when she lacks hard evidence? Although uncertainty prevents her from making reliable predictions, affect is based on predictions from existing knowledge and past experience (Barrett 2017, p. 78) and on the projection of scenarios revolving around futures worth avoiding or approaching. Since convincing an audience that departing from the status quo is worthwhile, or at least marginally better than inertia, the speaker may diagnose the ongoing present as worthy of action by describing how maintaining the status quo--which naturally stems from the past overlapping with the present--can lead to dismal futures worth avoiding (G'). Like loss-framing, such a diagnosis emphasizes the negative consequences of noncompliance (O'Keefe 2013, p. 123). Similarly, the speaker may emphasize how taking steps towards better futures worth attaining (G) depends on making this decision. Such depictions may then lead to appraisals of goal relevance, including concerns for the well-being of the decision maker, thus prompting experiences of emotions such as hope, fear, and anger (Moors et al. 2013). A key aspect is how a speaker then credibly gives the future presence.
3.2.1.1 Retrospective Forecasting I suggest that an argument by example works by invoking a known recent past, which then functions as an analogy of an anticipated near future worth either avoiding or approaching. Plantin argued that an analogy can help construct various types of feelings rhetorically-argumentatively and thus transfer emotions from the past to the present or an anticipated future because the analogous situations appear similar and within close proximity temporally and/or spatially (1999, p. 11-12). Perelman and Olbrechts-Tyteca distinguished between three approaches taken by a speaker seeking to establish a conclusion through a particular case: argument by example, illustration, and model/anti-model (1969, p. 350). Illustration is intended to increase adherence to a well-accepted rule, whereas example is aimed to establish a rule, temporally working by drawing on a particular case sufficiently probable to be one of general principle and thus helpful in avoiding or achieving future outcomes in the present case. I suggest labeling this mechanism retrospective forecasting, as it allows a speaker to give presence to what people in the invoked example did in a comparable case, but with the knowledge that currently exists in the situated present. Accordingly, such cases allow for both imitation and avoidance, thus warranting appeals to positive and negative consequences, respectively (Walton 2006, p. 106).
When we view an argument by example through a lens of retrospective forecasting, it is worth mentioning Quintilian's notion of 'vivid illustration'. Especially, such illustrative representations may function persuasively because of both their appeal to the imagination and ability to make a 'transference of time': "Nor is it only past or present actions which we may imagine: we may equally well present a picture of what is likely to happen or might have happened." (1922, p. 399). In emphasizing the imaginative (and hence temporal) aspect of vivid illustrations, Quintilian underlined the interaction between facts and how a decision maker (judge) feels when assessing them hereby mirroring the ongoing convergence between cognition and emotion that I have emphasized throughout this paper.
Such vivid illustrations enable a decision maker to "imagine to himself other details that the orator does not describe" (Quintilian 1922, p. 247) but equally important, in relation to the concept of retrospective forecasting, I suggest that the vivid projection of future (imagined) outcomes and goals, whether worth avoiding or approaching, draws its presence from existing cases; the more recent and more familiar, the greater impact. Such a transference of time may only provide answers about decision outcomes by virtue of being temporally situated in the crux between the past and the ongoing present, that is, by being temporally compared to the situated present in which a decision is to be made. Examples give credibility to an inherent claim about a future projection made by appraising aspects of certainty even though logical demonstration is futile. This might sound paradoxical when it comes to dealing with decision making under uncertainty. However, there is a point: if a speaker projects a future worth avoiding, but the scenario seems unconnected to existing phenomena and thus unrealistic to the audience, the credibility decreases, and the projection may cease to function as a vivid (and hence credible) future scenario worth avoiding. This is the fate of so-called empty threats, not only because the threatened consequences might not come about, but also because the causal mechanism appears either completely unlikely or is unknown to the audience.
In sum, the first dimension is to argue that a decision is necessary. To do so, I propose, a speaker must show how the exigence demanding a decision is temporally close, as in an imminent threat or a passing opportunity. The next task is to show the audience that outcomes are contingent on the proposed decision--in other words, that its decisions matter.
Mediators of Change (Must) Have Agency
To show the above contingency, the speaker must present reasons why the decision makers are "mediators of change," thus enabling them to acknowledge and accept that they possess the agency to actually affect the situation. Humans only deliberate about things within their power to change (Pfau 2007, p. 227;Kock 2017, p. 35). Accordingly, if an audience has no belief of such power, it will have no reason to care, in which case the speaker runs the risk of unwittingly convincing the audience to be utterly indifferent (lethargy) or give up before it even starts (despair). The speaker has to instill an agentic belief in the audience that it can cope and make a difference that leaves open an avenue of hope (Nussbaum 2018, p. 206).
Prospective Remembering
Another mechanism included in the model is the use of anticipated emotion to support the perception of the agency needed to make decisions in the present. I call this prospective remembering, which entails how it feels to be a person imagining herself situated in the future and looking back at the present in which she is to make her decision.
In general, decisions function as attempts to achieve positive future feelings, such as pride, and avoid negative emotions, such as guilt and regret (Lerner et al. 2015). Therefore, the anticipation of an emotion like regret can provide a reason to eschew excessive risk-taking. Notably, anticipated emotion does not appear to function independently of anticipatory emotions like fear and hope, just as the re-presentation of a past-oriented emotion like pride may support a presently experienced anticipatory emotion of hope (Cigada 2006), which in turn enables one to anticipate a future emotion of relief at overcoming a burdensome challenge.
Acting now in order to avoid feeling regret in the future can be a rational decision; that is, committing in the present to achieve or avoid the anticipated emotion related to future outcomes can indeed be rational. Although traditional economics textbooks have viewed sunk cost as a fallacy, the fallacious nature depends on whether one views rationality as utilitarian (cost-benefit) or deontological (commitment), leading to two distinct views of decision making; respectively a cost-benefit model and a model of practical reasoning and commitment (Walton 2002, p. 492). In short, if an actor bases a decision and action on a central personal principle (e.g. honesty), that act has value in and of itself regardless of a calculation of its consequences on a cost benefit scale because it helps her reason and navigate practical uncertainty "where exact calculation of costs and benefits is not possible, or would not be realistic." (Walton 2002, p. 494). In relation to the current conceptualization of a temporality of affecting argumentative action, Walton's point that precommitment can be a rational strategy (2002, p. 495) is helpful because it helps bridge understandings of practical reasoning as a process involving sunks costs (in the past) and appeals to anticipatory and anticipated (future) emotions such as pity and fear (Walton 1997(Walton , 2013. To be precise, emotions do not exist independent of the passing of time, and appeals to emotion (such as fallacious fear-appeals that rely on misinterpreted or false premises) gain their persuasiveness from how they evolve in light of new knowledge and experience.
Although certainty is a key difference between anticipatory (uncertain) and anticipated (certain) emotions (Baumgartner et al. 2008), anticipatory emotions experienced in the present may indeed directly relate to decisions and a pre-factual imagination of future states in which anticipated emotions arise. When decision makers make assumptions about the future occurrence of desired or undesired events and anticipate emotions, they still base these forecasts on both uncertain data and the potential contingencies of their own decisions. As such, fear might arise when one faces a dangerous threat like COVID-19, and uncertainty means that no one knows precisely how to avert disaster without jeopardizing democratic freedom. At the same time, however, one experiences a wide array of anticipated emotions, such as relief and joy, if the fear-inducing threat is successfully eliminated, and regret and disappointment if not. Similarly, anticipated emotions can help one stick to longterm goals by, for example, imagining future emotions of accomplishment.
Nonetheless, as with the mechanism of retrospective forecasting, which achieves a presence by establishing a past-future-present link, an emotional mechanism of prospective remembering still needs presence to affect a decision and, to invoke presence, a speaker must appeal to the audience's existing experiences (Tucker 2001). Therefore, prospective remembering also draws on the past, but in the reverse order, thus achieving presence by establishing a future-past-present link. A speaker must draw on existing experiences and values from the past to enable decision makers to imagine how it feels to regret a present failure to make a decision that could have precluded undesirable consequences.
Argumentative Action
Third, despite the constraints of a present situation, a belief in the contingencies of one's decision is insufficient. As such, Pfau (2007, p. 224) applied the virtue of courage-which lies between the extremes of fear and confidence-to explain how an audience might move from being inclined to have sufficient confidence to actually 1 3 making a decision. This movement from civic fear to contingency and a confidence to act on the arguments presented echoes Nussbaum's (2018) point that faith must bolster hope to be worthwhile. She says that if we think "our efforts are a waste of time, we don't embrace hope" (p. 214). The connection between hope and faith illuminates how faith relates not only to the emotion of hope, but also to aspects of confidence and processes of trust (Khodyakov 2007). Temporally, the dimension of faith is past-oriented, gathering its reasons from past events in order to qualify whether there is reason to believe in the advocated course of action.
Positive anticipatory emotions like hope rely on some degree of belief that one's decision (D) might enable better outcomes (G) than if one refrained (D') from engaging in a given activity involving a worse outcome (G'), all of which again reflects decision makers' appraisals of agency and coping potential. Nussbaum wrote: "We need to believe that the good things we hope for have a realistic chance of being realized through the efforts of flawed human beings" (2018, p. 213).
Thus, the synthesis of temporality and emotion in argumentation that adheres to a suggested proposal in the present transcends the temporal constraints of uncertainty. Such a commitment arises both because emotions experienced in relation to past events are re-interpreted and because emotions that may arise at future events are re-imagined. Scott (2020) underlined how adherence exists because of its relation to the past and future: On the side of the past, what we presently adhere to can be understood as a kind of personal precedent, as the past weighing on the present as a constraint on what we will consider to be argumentatively reasonable (from myself and from others). On the side of the future, we will find that adherence makes reference to a number of possible futures where, under certain conditions, we would be committed to acting in certain ways given our current configuration of value commitments. (Scott 2020, p. 31).
To this, I should add that such adherence depends on the present emotional experience, which stems from the negotiation of how emotion constitutes the willingness to decide under uncertainty.
COVID-19: It Is Better to Act Today Than Regret Tomorrow
I now use the conceptualized model to illustrate how Mette Frederiksen on March 11, 2020, portrayed two possible scenarios to show her reasoning in support of her proposal to the Danish population to practice physical distancing.
During her speech, Frederiksen introduced what became a familiar catchphrase of the Danish coronavirus response: "Now we must stand together by keeping a distance." In this instance, a "principle of caution" underlays the main practical inference (Walton 2006, p. 300), in which the goal was to protect "the most vulnerable people in our society" (Frederiksen 2020a). She presented the action of physical distancing as the means of slowing the spread of the virus and thus realizing this goal. Indeed, she emphasized the need to take action today in order to avoid regret in the future: It is better to act today than regret tomorrow. We must take action where it has an effect. Where the disease is spreading [...] Therefore, the authorities recommend that we shut down all unnecessary activity in those areas for a period. We are adopting a principle of caution. While Frederiksen's argument rests on acceptable scientific knowledge, four days after the March 11 press conference, she underlined that the decision to lock down much of Danish society was ultimately political: "If I have to wait for evidence for everything in handling the coronavirus, then I am certain we will be too late." (Frederiksen 2020b). Although the science says that close physical contact spreads the disease, the consequences of mandating a societal lockdown to avoid such contact are far more political in the sense that "any action that promotes one good or value tends to counteract others" (Kock 2017, p. 58). Frederiksen stressed: "We must minimize activity as much as possible. But without bringing Denmark to a halt. We must not throw Denmark into an economic crisis" (2020a).
Using the developed argument model (Fig. 1), I can show how Frederiksen constructed two decisions: either citizens decide to follow and support the recommended proposal of physical distancing, D, leading to a desirable future state of flattening the curve, G, or they do not distance, D', which will lead to an undesirable future state worth avoiding at almost any cost, G'. The movement from D to G appears consequential despite the uncertainty of a novel disease. Equally important from a temporal and emotional perspective, an allusion to the distant and recent past makes the consequences of deciding to show public spirit and comply with physical distancing more credible, while the argumentative force of the outlined consequences depends on the emotions they invoke (see Fig. 2 "Italy is locked down. In the hospitals, there is a need for respirators and personnel. I would like to underline: It is not a scare story. It is not a fancifully conceived future scenario. It is the reality in a country that most of us know." Physical contact and increase in the spread of disease (D') "We have moved towards the next phase of the epidemic, in which the spread of disease not only comes from travelers. But where we have started to infect each other in Denmark." Fear of uncontrollable epidemic (G')
RetrospecƟve forecasƟng
" We must avoid too many people becoming infected at the same Ɵme. As it has happened in Italy."
Fig. 2 An illustration of the temporality of affecting argumentative action
In the following sections, I detail how Frederiksen sought to connect the threat that COVID-19 posed, while also instilling a degree of belief that following government guidelines could make a difference. As such, she established the threat as contingent and invoked an element of courage that spurred decisive readiness.
Exigence
Frederiksen sought to establish the danger of COVID-19 and demanded action at a time when global news stories abounded and the disease was becoming serious in Denmark, but as of March 11, any Dane infected with the virus had yet to die (Sundhedsstyrelsen 2020).
When I stood here yesterday, there were 157 Danes infected with corona. Today, we have 514. That is more than a tenfold increase since Monday, where it was 35. The coronavirus spreads extremely fast.
The rapid increase in cases supported Frederiksen's claim that the disease was not only dangerous but also spreading swiftly through Danish society, bringing an inevitable future threat ever closer. Urgent action was required, with the accent on urgent.
At the press conference, Frederiksen used Italy as an argument by example, stressing what Denmark should avoid. The Italian example enabled her to use the temporal mechanism of retrospective forecasting by drawing on the known recent past as an analogy for an anticipated near future worth avoiding and therefore as a present reason for physical distancing. Interestingly, Frederiksen rebutted a potential objection that the Italy reference was a scare example, emphasizing its "reality." In doing so, she defined a scare example as a "fancifully conceived future scenario," stressing that in contrast to the recent past, Italy served as a real example, one that could warn a Danish audience of the possible future consequences of present inaction against COVID-19. In the week leading up to her March 11 press conference, the Italian government had placed several of its northern provinces under lockdown, and on March 11 the cumulative death toll in Italy reached 827 (Remuzzi and Remuzzi 2020). In this context, Italy served as a well-grounded example capable of warning and potentially scaring a Danish audience because Danes know the country and can thus more easily accept the comparison as relevant and worth avoiding.
Contingency
While the numbers of infected citizens and the speed with which the virus was spreading could indeed support the severity of the situation, the target deemed dangerous and therefore worthy of fear could not be so overwhelming as to cause people to believe that no matter what they did, the crisis would strike (Pfau 2007). Frederiksen tried to inspire confidence in the potential of action by emphasizing that citizens should act in the present instead of waiting and regretting their inaction, underlining that physical distancing is precisely the measure to hinder the virus in spreading.
Although regret is a past-oriented emotion, Frederiksen contrasted taking action now (present) with a prospective remembering of regret. Although regret may stem from both following non-beneficial advice and ignoring beneficial advice (Tzini and Jain 2018), Frederiksen's appeal to act in order to prevent a future feeling of regret draws its argumentative force from the certainty of physical distancing vis-à-vis the uncertainty of inaction, thus leading to an anticipated regret of how it generally feels to ignore the certainty of beneficial advice. In sum, in this instance adherence depended on an inference stating that sacrificing present freedom was worthwhile to avoid a greater future loss, such as life itself. One can view Frederiksen as attempting to bridge the uncertainty of navigating a "situation that does not look like anything we have tried before" with the certainty of anticipated regret, as this quote illustrates: "But the alternative-not to do anything-would be far worse. I hope there will be an understanding for that. I am convinced that there will be." In addition to regret, Frederiksen emphasized the opportunity for agency that lay ahead and reinforced such statements by highlighting what was already taking place in the recent past and ongoing present: We must help each other. Show strength-think about others. Especially about those who are vulnerable. I would like to thank everyone in our health sector for the great contribution you are making. Thank you for your contribution now. And thank you in advance for your contribution in the coming days, weeks, and months. I am going to tell it like is. It is going to be tough. This situation puts great demands on all of us.
By speaking directly to essential workers, who were far more exposed than other parts of the population that could work from home or had been sent home, Frederiksen acknowledged both the work taking place and what lay ahead.
Confidence to Act
Lastly, while decision makers (e.g., healthcare professionals) must acknowledge the unfolding of events as contingent on their own actions, one needs the confidence to act to avoid the paralysis of what could be labeled well-informed hopelessness. Despite the "extraordinary situation," Frederiksen encouraged citizens to stand up for Danish values when it mattered, underlining the goals of acting with an eye to the common good: "Let us now show what we are capable of when it matters. The Danes are already at it. We are showing public spirit. That is what works." By emphasizing what was already taking place (drawing on the recent past and ongoing present), Frederiksen stressed that agency and coping potential ("a huge responsibility") were possible if one transcended the future and past into the present. While "proving" the future is inherently impossible in argumentation (Perelman and Olbrechts-Tyteca 2020), adherence to a proposal of, say, physical distancing, as Frederiksen advocated, depends on whether there are any compelling reasons to believe the future worth achieving will be realized (Nussbaum 2018). Ongoing action from civil society, drawing on a legacy of public spirit, may well have increased the felt probability of success in protecting the weakest citizens, even though predictions for specific measures were unreliable.
To summarize, I have illustrated how Mette Frederiksen, sought to gain support for her proposal to maintain physical distancing as a means of stopping the spread of COVID-19. Above all emphasizing negative future consequences worth avoiding, she translated these futures into the present by drawing on both the recent past (the Italian experience and lack of decisiveness) as an argument by example and by addressing the need to act now in order to avoid a future feeling of regret.
Conclusion: Taking a Leap of Faith
On March 11 2020, the W.H.O. declared COVID-19 a global pandemic, and Danish Prime Minister Mette Frederiksen told the Danish population that this would have serious ramifications in the near future. Globally, the consequences of the pandemic have varied greatly, in terms of both fatalities and restrictions on freedoms. Two pertinent questions concern, first, the speed with which different governments responded and, second, the reasoning government leaders of democratic societies applied to their preemptive proposals aimed at mitigating the yet unseen consequences.
To understand how such argumentation under uncertainty functions, this study has combined two strands of theorizing within the argumentation literature: temporality and emotion. Starting from the premise that rhetorical argumentation is practical reasoning about choice of action, I have argued that in situations of uncertainty argumentation affects action, such as decisions, via appeals that invoke emotion and thereby translate the distant past and future into the situated present. Building on a dynamic understanding of emotion as having not only cognitive effects but also cognitive origins, I have suggested a model of affecting argumentative action and identified two intertemporal mechanisms-retrospective forecasting and prospective remembering-as a means of explaining how the distinct temporality of emotion enables argumentative action. For instance, an argument by example functions persuasively in situations marked by high uncertainty through the emotional analogy it makes. This does not happen because an example provides full epistemic certainty about future consequences, but rather because it minimizes the gap between an epistemic waiting game for certainty and a prudential willingness to act incisively, thus allowing a decision maker to commit herself and take the leap of reasonable faith that is a defining characteristic of human choice. | 2021-01-14T14:27:37.690Z | 2021-01-13T00:00:00.000 | {
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25603840 | pes2o/s2orc | v3-fos-license | Thickness of cumulus cell layer is a significant factor in meiotic competence of buffalo oocytes.
This study evaluated the meiotic competence of buffalo oocytes with different layers of cumulus cells. A total of 588 oocytes were collected from 775 ovaries averaging 0.78 oocytes per ovary. Oocytes with homogenous cytoplasm (n = 441) were selected for in vitro maturation (IVM) and divided into four groups based on their cumulus morphology: a) oocytes with ≥ = 3 layers of cumulus cells, b) 1-2 layers of cumulus cells and oocytes with partial remnants or no cumulus cells to be cocultured c) with or d) without cumulus cells. Oocytes in all four groups were matured in 100 μL drop of TCM-199 supplemented with 10 μg/mL follicle stimulating hormone (FSH), 10 μg/mL luteinizing hormone (LH), 1.5 μg/mL estradiol, 75 μg/mL streptomycin, 100 IU/mL penicillin, 10 mM Hepes and 10% FBS at 39C and 5% CO2 for 24 hours. After IVM, cumulus cells were removed from oocytes using 3 mg/mL hyaluronidase, fixed in 3% glutaraldehyde, stained with DAPI and evaluated for meiotic competence. The oocytes with ≥ = 3 layers of cumulus cells showed higher maturation rates (p < 0.05: 64.5%) than oocytes with partial or no cumulus cells (8.6%) and oocytes co-cultured with cumulus cells (34.5%) but did not differ from oocytes having 1-2 layers of cumulus cells (51.4%). The degeneration rates were higher (p < 0.05) for oocytes with partial or no cumulus cells (51%) than rest of the groups (range: 13.8% to 17.4%). These results suggest that buffalo oocytes with intact layers of cumulus cells show better IVM rates than oocytes without cumulus cells and the co-culture of poor quality oocytes with cumulus cells improves their meiotic competence.
Introduction
The successful in vitro maturation, fertilization, and culture (IVM/IVF/IVC) of bovine oocytes has brought interest to implement this technique in water buffalo (Bubalis bubalis) for in vitro production of embryos. The inherent problem of low oocyte yields from buffalo ovary [8,11,27,32] makes the use of IVF procedures questionable given the cost of IVF and low oocyte yield. This poor oocyte yield has been attributed to low number of primordial follicles (10,000 to 19,000) in the buffalo ovary [10,26] compared to 150,000 in cattle [14]. Despite this major factor, high rates of 70-90% for IVM [4,8,17,22], 60-70% for IVF [8,17,21,33] and 40-50% for cleavage rate [5,17,20,21] have been observed. However, blastocyst development is still very poor and ranges between 10-30% [3,4,6,21,23]. The number and quality of oocytes further decreases during summer months [22,29] allowing fewer oocytes available for IVF studies. Considering the low yield of oocytes from the buffalo ovary, this investigation was carried out to utilize all the available oocytes with homogenous cytoplasm with varying layers of cumulus cells for IVM.
Materials and Methods
This study was conducted during the months of July and August when minimum temperature varied between 27 o C (80.6 o F) to 32 o C (89.6 o F) and maximum temperature varied between 31 o C (87.8 o F) to 37 o C (98.6 o F). The relative humidity varied between 66 to 89% during trial period. Unless specified, the reagents were from Sigma Chemicals (St. Louis, MO, USA).
Collection of oocytes
Ovaries from adult buffalos of Nili-Ravi breed were collected immediately after slaughter and transported to the laboratory within two hours of collection in an insulated container at 35-37 o C. Upon arrival, ovaries were washed twice with normal saline containing 100 IU/mL of penicillin *Corresponding author Phone: +1-801-587-3706; Fax: +1-801-581-6127 E-mail: kazimchohan@hotmail.com and 100 µg/mL of streptomycin. Oocytes were aspirated from 2-8 mm follicles with an 18-gauge needle fitted to a 12 mL disposable syringe and transferred to 15 mL polystyrene centrifuge tubes in a 37 o C water bath. Oocytes were recovered from the settled sediment after 15-20 minutes using a low power (20X) stereomicroscope. The data regarding number of ovaries used, number of oocytes collected, quality of the oocytes and the number of oocytes used in each replicate was recorded.
In vitro maturation of oocytes
Oocytes with homogenous cytoplasm were divided into four groups based on their cumulus morphology: a) oocytes with ≥ = 3 layers of cumulus cells, b) 1-2 layers of cumulus cells and oocytes with partial remnants or no cumulus cells to be co-cultured c) with or d) without cumulus cells. Oocytes in all four groups were washed thrice in medium 199 and transferred to 100 µL droplets (5-8 oocytes/drop) of maturation medium (TCM-199: Gibco Life Technologies, NY, USA) under sterile mineral oil in plastic dishes and incubated at 39 o C and 5% CO 2 in air for 24 hours. The maturation medium was supplemented with 10 µg/mL FSH, 10 µg/mL LH, 1.5 µg/mL estradiol, 75 µg/mL streptomycin, 100 IU/mL penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS: Hyclone Laboratories Inc. Logan, UT, USA). Freshly detached cumulus cells were washed twice by centrifugation at 300 g in maturation medium and 1-1.2 × 10 6 /mL cells were added to each microdrop of maturation medium in the co-culture group.
Evaluation for meiotic development
After IVM, oocytes in all groups were exposed to 3 mg/ mL hyaluronidase in saline and cumulus cells were removed by repeated pipetting. Denuded oocytes were fixed in 3% glutaraldehyde in saline at room temperature for 15 minutes, rinsed and incubated in 0.001% 4, 6 diamidoino-2phenylindole (DAPI), a fluorescent stain specific for nuclear material for 20 minutes at room temperature. Oocytes were rinsed in saline to remove DAPI particles, mounted on glass slides, and evaluated for meiotic development (oocytes reaching metaphase-II) at 400x using a Nikon microscope equipped with fluorescent illumination and filters giving maximum transmittance at 405 nm. An oocyte was classified as degenerated if the nuclear material was scattered in the ooplasm indicating spindle damage.
Statistical analysis
Data for meiotic development of oocytes among all groups was analyzed by chi square procedure using Statistix Analytical Software, Tallahassee, FL, USA.
Results
A total of 588 oocytes were aspirated from 755 ovaries in nine replicates of which 441 oocytes with homogenous cytoplasm and varying layers of cumulus cells were used for IVM while the remaining were used for cumulus cells or discarded due to poor quality. The number of oocytes collected averaged 0.78 per ovary ( Table 1). The IVM rates for oocytes with ≥ = 3 layers (64.5%) and 1-2 layers of cumulus cells (51.4%) were significantly (p < 0.05) higher than oocytes with partial remnants or without cumulus cells (8.6%) and oocytes co-cultured with cumulus cells (34.5%). No difference was observed for IVM rates between oocytes having ≥ = 3 and 1-2 layers of cumulus cells. The degeneration rates were higher (p < 0.05) for oocytes without cumulus cells (51%) than all the other groups (Table 2).
Discussion
Cumulus cells have been considered to play an important role in oocyte maturation by keeping the oocyte under meiotic arrest, inducing meiotic resumption and by supporting cytoplasmic maturation. These functions have been attributed to their gap junctions and their specific metabolizing capabilities [31]. Physical contact between oocyte and cumulus cells has been considered necessary for the transfer of nutrients and factors essential for oocyte development [2]. However, dissociated cumulus cells have been reported to produce paracrine factors, which resume meiosis in denuded oocytes [13]. The results of this study showed that buffalo oocytes with homogenous cytoplasm surrounded by compact layers of cumulus cells had a significantly higher maturation rate than oocytes with partial remnants or no cumulus cells matured with or without additional cumulus cells. The modest increase in IVM rates of co-cultured oocytes in this study can be justified by the fact that paracrine factors produced by the added cumulus cells might have been only transferred to partially denuded oocytes via available gap junctions whereas such communication seems absent in similar oocytes matured without cumulus cells. This is also evident from the results that addition of cumulus cells not only improved IVM but also rescued oocytes from degeneration whereas more oocytes without somatic cell support underwent degeneration. Present study yielded 0.78 oocytes per ovary, which is low compared to previous findings of 1.49 [8] and 1.76 [27] for the same breed of buffalo. This difference in oocyte yield is due to seasonal variation because the other studies were conducted during the cooler months of winter. Fewer follicles were found on buffalo ovaries at slaughter during summer than winter months [25] and buffaloes under heat stress produced fewer good quality oocytes than unstressed buffaloes [29]. The number of oocytes decreased from 1.7 to 0.9 [11] and from 0.7 to 0.4 [32] per ovary when selected for IVM on the basis of cumulus morphology. These findings are in agreement to cumulative number of 0.42 oocytes per ovary recovered with ≥ = 3 and 1-2 layers of cumulus cells in present study. Datta and Goswami [9] observed a significant drop from 1.02 to 0.84 in oocyte yield and a decrease from 0.21 to 0.14, 0.45 to 0.33 and 0.46 to 0.35 for good, average and poor quality oocytes per ovary in buffalo when temperatures increased from <25 o C to >25 o . Nandi et al. [22] also found a decline from 1.22 to 0.85 oocytes per ovary when oocytes were collected during cool (1-10 o C) and hot (> 30 o C) months, respectively. Their IVM rates also differed between cool (89%) and hot (72%) seasons but no difference was observed for fertilization, cleavage and blastocyst development because only matured oocytes were used for IVF/IVC. A marked decrease in oocyte yield, quality and developmental ability has been also reported in Bos taurus cows [24].
Our IVM rates for oocytes with ≥ = 3 and 1-2 layers of cumulus cells are lower than IVM rates of 84-91% previously reported for the same breed of buffalo [8]. This difference is due to season as well as the use of quality oocytes. In a previous study, Chauhan et al. [4] found significantly different IVM rates of 85, 54, and 26% for grade 1 (≥ = 5 layers of cumulus cells and homogenous cytoplasm), grade 2 (≤ = 4 layers of cumulus cells and homogenous cytoplasm) and grade 3 (without cumulus cells and irregular shrunken cytoplasm) buffalo oocytes. The subsequent fertilization, cleavage and blastocyst rates were also different in relation to the quality of the oocytes and no blastocyst was formed from grade 3 oocytes. They suggested that the embryo yield can be predicted after IVMFC by gross morphological appearance of the aspirated oocytes and grade 2 oocytes can be effectively used in buffalo IVF system however their paper lacks the information about the season of study which is a critical factor in buffalo reproduction. Similar observations were recorded for good, fair, and poor quality oocytes in Egyptian buffaloes [1]. Though buffaloes cycle throughout the year, they show a very significant seasonality in breeding that only 4% come into estrus from April through July [18]. Considering the low availability of quality oocytes from buffalo ovary, attempts have been made to utilize the oocytes recovered in denuded form during aspiration in an IVF system. In a previous study [12], addition of cumulus cells in maturation medium restored the nuclear maturation of artificially denuded oocytes (64%) close to the compact cumulus enclosed oocytes (66%) but oocytes recovered in denuded form (46%) never reached the same levels of IVM. In another study [30], buffalo oocytes with compact and dense cumulus cells showed higher IVM rate (p < 0.01: 67.3%) than oocytes with thin cumulus layer (27.5%) or with small remnants of cumulus cells and poor naked oocytes (3%). These findings are in agreement to present results but again lack the information about season of study.
Lower IVM and IVF rates have been also reported for cumulus free oocytes compared to cumulus enclosed oocytes in cattle [7,15,19,28,34]. In summary, the results of present study suggest that nuclear maturation can be restored in a substantial number of buffalo oocytes recovered in denuded form or with partial remnants of cumulus cells by addition of cumulus cells in maturation medium. However, further studies are required to understand the cytoplasmic maturation of such oocytes, which is essential for male pronucleus formation and subsequent embryonic development. We also suggest that IVM/IVF/IVC procedures in water buffalo should be preferably performed during the cool months of winter and season of experiment be reported in publications. Our understanding is that the studies on abattoir ovaries may not be truly representative of the potential of the buffalo ovary as mostly aged, underfed and post lactation animals are slaughtered. Therefore, further studies should be focused on IVMFC of oocytes recovered by ultrasound guided transvaginal aspiration from young live animals in good body condition. | 2018-04-03T03:55:39.610Z | 2004-09-01T00:00:00.000 | {
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271243038 | pes2o/s2orc | v3-fos-license | A dataset of topographic correction coefficients for shortwave downward radiation over the Pan-Third Pole
Prevalent Shortwave downward radiation (SWDR) estimates assume a flat surface, neglecting topographic effects and leading to significant errors in mountainous regions. We introduce SWDR topography correction coefficients (TCCs), based on the mountain radiative transfer model tailored for the Pan-Third Pole region. This dataset effectively bridges the disparities between flat-surface SWDR and rugged-surface SWDR, forming part of the Long-term Earth System spatiotemporally Seamless Radiation budget dataset (LessRad). Validation results using a three-dimensional radiative transfer model demonstrate the efficacy of this method in correcting solar direct radiation, sky diffuse radiation, and SWDR under diverse conditions. At a spatial resolution of 2.5 arc-minutes, the correction accuracy for solar direct radiation is characterized by a coefficient of determination (R²) of 0.998, a relative root mean square error (rRMSE) of 2.4%, and a relative bias (rbias) of 0.8%. For sky diffused radiation, an R² of 0.965, a rRMSE of 1.2%, and a rbias of −0.8%. SWDR corrections under clear and cloudy skies also show high accuracy, demonstrating the robustness of the TCCs approach.
in SWDR estimations within mountainous regions, and inappropriately smoothing the spatial distribution of SWDR 15,16 .Mountainous regions exhibit significant spatiotemporal heterogeneity in SWDR, influenced by factors such as slope, aspect, and shading effects 17,18 .Existing topographic correction methods of SWDR in mountainous terrains primarily fall into two categories: mountain radiative transfer models (MRTM) 15,16,[19][20][21][22] and machine learning approaches [23][24][25][26] .Among these, MRTM describes the complex interaction between SWDR and the surface, facilitating the derivation of SWDR values actually received by mountainous terrain.In contrast to machine learning, MRTM offers an advantageous combination of specific physical principles and high terrain correction accuracy, albeit at the cost of computational intensity and operational inefficiency 27 .The utilization of MRTM for terrain correction of planar-assumed SWDR necessitates a sequence of scale conversion procedures.Specifically, MRTM is devised at the Digital Elevation Model (DEM) scale, typically with a spatial resolution of meter-level, while common SWDR datasets boast spatial resolutions of kilometer-level.Consequently, it becomes imperative to initially downscale the SWDR outcomes to match the spatial resolution of the DEM, employing techniques such as resampling, to drive the MRTM for terrain correction.Subsequently, these downscaled results need to be re-aggregated to their original kilometer-level spatial resolution.The scale conversion process demands substantial computing resources and is notably time-intensive.Moreover, we have observed ongoing optimization efforts in methods for estimating SWDR, particularly enhancing accuracy on flat surfaces.Simultaneously, the correlation between SWDR under assumption of a flat surface and it received on actual mountainous surfaces is primarily contingent on terrain characteristics and remains unaffected by alterations in data sources and inversion methodologies.Consequently, we propose the Topography Correction Coefficients (TCCs) dataset as a pivotal intermediary to facilitate the application of various SWDR results inverted under the assumption of a flat surface in mountainous regions.And it serves as a topography correction coefficients product within the Long-term Earth System spatiotemporally Seamless Radiation budget dataset (LessRad).
In the Pan-Third Pole region, with the Tibetan Plateau as its center, stands as one of the world's most complex terrestrial environments, significant terrain variation has profound effects on SWDR 28 .To facilitate effective SWDR terrain correction within this intricate region using MRTM, this study has introduced a multi-spatial scale terrain correction parameterization product based on Shuttle Radar Topography Mission (SRTM) data.The SRTM, conducted by NASA, aimed to obtain high-precision three-dimensional terrain data of the Earth's surface using radar imaging technology deployed on the space shuttle.This parameterization dataset covers spatial resolutions of 3 arc-seconds, 15 arc-seconds, 30 arc-seconds, 1 arc-minute, and 2.5 arc-minutes.It comprehensively addresses the impact of varying solar positions and terrain conditions on SWDR, encompassing direct radiation and sky diffuse radiation.The result is a transformative process that converts original SWDR obtained under the flat surface assumption into the terrain-corrected SWDR considered the effect of mountainous surfaces.
The accuracy of terrain-corrected SWDR depends on two critical factors: the chosen terrain correction method and the accuracy of the original SWDR data assumed under the flat surface.Several considerations prompted our decision to construct a comprehensive SWDR terrain correction coefficient dataset, rather than directly providing SWDR results after terrain correction.(1) With the continual advancement of remote sensing technology and inversion algorithms, there are improvements in spatial and temporal resolutions of SWDR products, as well as their accuracy.(2) MRTM have showed exceptional accuracy in SWDR correction.Simultaneously, the accuracy of Digital Elevation Model (DEM) data remains reliably stable, with minor improvements in DEM data accuracy having minimal influence on the outcomes of terrain correction.(3) In contrast to the variety of available SWDR products, terrain-corrected SWDR products remain notably scarce.The SWDR terrain correction parameterization defined in this study is established as the ratio between SWDR received by the rugged surface and SWDR received under the assumption of a flat plane.This ratio is inherently linked to the terrain characteristics and remains unchanged regardless of variations in incident radiation magnitude.Consequently, for SWDR products obtained under the flat surface assumption, at any given moment and through any method, such SWDR terrain correction coefficient products serve as a versatile tool for efficient terrain correction implementation.By constructing multi-spatial scale TCCs dataset, we streamline the terrain correction process for data users, enabling them to select TCCs results with the same spatial resolution as the SWDR data.Through simple processing, users can obtain terrain-corrected SWDR results in mountainous areas.This approach not only ensures reliable results but also significantly reduces the computational burden on data users.Our overarching goal is to offer support to current and future SWDR researchers in mitigating the uncertainties arising from terrain effects when applying their algorithms to mountainous regions.This TCCs datasets are not only applicable to various existing SWDR datasets without terrain correction but also to potentially higher-precision SWDR results under future flat surface assumptions.
Methods
Study area.This study primarily focuses on the Tibetan Plateau and its neighbouring regions, located within the geographic coordinates of 20°-50°N and 60°-110°E, encompassing Central Asia-an area colloquially referred to as the Pan-Third Pole (Fig. 1).This extensive region holds the distinction of being the largest expanse of the glacier and snow-covered terrain outside the polar regions.It serves as a critical reservoir of freshwater resources and is renowned as the ' Asia Water Tower' 29,30 .This geographical feature plays a pivotal role in the context of global climate dynamics, carbon equilibrium, biodiversity preservation, and various other ecological factors [31][32][33][34] .Simultaneously, its intricate and multifaceted surface morphology exerts a profound influence on the spatial distribution of SWDR at the Earth surface 15,16 .To better evaluate our research, we analyzed the slope characteristics in the Pan-Third Pole region and identified the five 1°x1° areas with the highest average slope as sample demonstration areas, as outlined in Fig. 1 and Table 1.
Source data.The Shuttle Radar Topography Mission (SRTM) is a collaborative undertaking involving the National Aeronautics and Space Administration (NASA), the National Geospatial-Intelligence Agency (NGA), and the German and Italian Space Agencies.Its primary objective was to acquire high-resolution terrain data concerning the Earth surface.SRTM successfully generated a digital elevation model (DEM) covering a substantial portion of the Earth's surface, ranging from 60°N to 56°S.The SRTM DEM dataset is distinguished by its data accuracy and offers two key resolutions: SRTM1, featuring a horizontal resolution of 1 arc-second, and SRTM3, with a horizontal resolution of 3 arc-seconds [35][36][37] .For the Pan-Third Pole region, SRTM1 DEM data was obtained through the Google Earth Engine (GEE) platform 35,38,39 .Furthermore, calculations of slope and aspect were conducted and corresponding datasets were acquired through the GEE platform.
Method flow.This study builds upon the Uniform ShortWave Topographic Radiation Model (USWTRM) method, established by Xian 16 , and undertakes a comprehensive and detailed investigation of the terrain coefficient calculation process.The flow chart for obtaining terrain correction coefficient data for surface SWDR in the Pan-Third Pole region is depicted in Fig. 2.
The calculation of sky visibility factor.The Sky Visibility Factor (SVF) serves as a valuable metric for quantifying the proportion of the visible sky within the hemispheric space 40 , and it can be expressed as Eq.(1).
SVF c os Slope sin H sin Slope cos
where, H φ denotes the horizon angle for each direction φ.If there is no horizontal occlusion in direction φ, This coefficient holds significant importance in assessing the degree of openness or exposure of a given area to the sky and is particularly critical for the accurate quantification of diffuse radiation received by mountainous terrains.To achieve this, we employ DEM data to accurately determine the SVF for each pixel on the mountainous surface through a Monte Carlo simulation methodology.Specifically, our approach uses the target pixel as the central reference point, and systematically evaluating the occlusion of the target pixel within the visible sky range by neighbouring pixels at 16 azimuth angles.This process allows for the accurate computation of the SVF.It is worth noting that in the estimation of SVF using the Monte Carlo method, the selection of the search radii is a pivotal parameter, closely linked to the overall accuracy of the results 41 .To identify the search radii, we tested the SVF results under different search radii in five sample areas with the largest average slopes in Fig. 1.
Following tests in five representative sample areas (Fig. 3), we ultimately determined the search radii to be 3 km.It is worth noting that the 3 km distance is derived by converting the DEM data from the geographical coordinate system to the projected coordinate system.In the geographical coordinate system, this distance is expressed as 100 arc-seconds.This selection offers a favourable balance between computational efficiency and result accuracy.Moreover, while longer search radii allow for the consideration of potential occlusions by more Fig. 2 Flow chart of SWDR topography correction coefficient dataset.Where, the SZA is solar zenith angle; the SAA is solar azimuth angle; the SVF is the sky visibility factor; the cosSIA is the cosine of the solar incident angle; the TCCSDR is terrain correction coefficient for solar direct radiation; the TCCDIF is terrain correction coefficient for diffuse radiation; the TCCREF is terrain correction coefficient for adjacent surface reflected radiation.
Fig. 3 The average SVF calculated by using different search radius in Monte Carlo simulation.distant mountains, they also entail stronger path absorption and scattering of radiation by the atmosphere during transmission.Consequently, radiation changes induced by distant mountain occlusions become more susceptible to atmospheric influence, thereby introducing additional uncertainty.Furthermore, these search radii align with the prevailing spatial resolution of commonly used SWDR algorithms 6,12,42,43 , with a spatial resolution of around 5 km.As such, the selected search radii comprehensively capture the influence of local topography on the sky's visible range within each coarse pixel.
The calculation of shadow.Direct radiation uniformly illuminates all points on a flat surface, but in mountainous areas, it is highly susceptible to shadows caused by terrain obstacles.These shading phenomena encompass two primary categories: self-shading and shading induced by neighbouring mountains.To address this, we employ the Monte Carlo simulation method to calculate mountain shadows under varying solar zenith angles (SZA) and solar azimuth angles (SAA).Similar to the SVF, the calculation outcomes for shadow are influenced by the chosen search radii 44 .Therefore, we employ the same search radii as used for SVF.In the simulation, SZAs and SAAs are systematically sampled at 5° intervals.This approach yields shadow results for a range of SAZ and SAA combinations, comprehensively capturing the diverse scenarios of shading across mountainous landscapes.
The calculation of solar incidence angle.The solar direct radiation reaching the surface of mountainous terrain is governed by the solar incident angle.This angle is conventionally defined as the angle between the incoming solar rays and the perpendicular line to the slope.It is frequently expressed in the form of its cosine (denoted as cosSIA).Smaller solar incident angles, corresponding to larger values of cosSIA, result in a higher intensity of solar direct radiation per unit area received by the Earth's surface.The cosSIA is intricately linked to factors such as slope, aspect, SZA, and SAA.Its specific calculation formula is as follows: when cosSIA is greater than 0, it indicates that the Earth's surface is receiving solar direct radiation.Conversely, when cosSIA equals 0, it signifies that the ground surface is not receiving solar direct radiation due to self-shading or because the sun is positioned below the horizon.
The calculation of rugged surface area.Undulating terrains within mountainous regions often result in a surface area that is substantially larger than its projected area.This discrepancy in surface area has a direct impact on the irradiance (measured in W/m²) received by the mountainous surface.To calculate the rugged surface area for an DEM pixel, we utilize data from eight neighbouring DEM pixels.Specifically, this process involves the creation of eight irregular triangles (labelled I to VIII) connecting the central point of the target DEM pixel with the central points of the surrounding eight DEM pixels.Subsequently, the area of each of these triangles is calculated, and the sum of the areas falling within the domain of the target DEM pixel is determined 45 (Fig. 4).Its calculation formulas are as follows: S p represents the surface area of the target DEM pixel p, which is comprised of the area of eight triangles formed by the connection of the target pixel with eight neighbouring DEM pixels.
As an example, the area of the three-dimensional S I , formed by the p, t 1 , and t 2 , is determined through the utilization of Heron's Formula: Where, H p , H p1 , and H p2 are the elevation of DEM pixel p, p1 and p2, respectively.And S h is the projected area of the DEM pixel.
The calculation of area ratio.
To quantify the distinction between the actual surface area of mountainous regions and their projected planar area, we employ the area ratio factor (t) for measurement.
In theory, t should not exceed 1.However, in regions characterized by extremely steep terrains, significant slopes result in slope cos( ) approaching 0. Under such circumstances, even minor inaccuracies in slope calcula- tions can disproportionately impact t potentially leading to anomalous situations where > t 1.To mitigate the influence of outliers and reduce the error, we constrain ∈ t (0,1] (Eq. 11).
The calculation of terrain correction coefficient.After building upon the terrain calculations described above, we are equipped to compute the terrain correction coefficients (TCCs) at a 1 arc-second resolution for the three components of SWDR, namely: TCCSDR (Terrain Correction Coefficient for Solar Direct Radiation), TCCDIF (Terrain Correction Coefficient for Sky Diffuse Radiation), and TCCREF (Terrain Correction Coefficient for Adjacent Surface Reflected Radiation) 15,16,[19][20][21][22] .
In mountainous areas, solar direct radiation is influenced by factors such as the solar zenith angle, solar incident angle, shadow, and area ratio 16,21 .Consequently, the TCCSDR can be calculated using Formula 12.And the SVF represents the proportion of the sky hemisphere observed by surface pixels in mountainous regions.Hence, under the single slope assumption, SVF serves as a conversion coefficient for the sky diffuse radiation received by mountainous surfaces compared to flat surfaces 16,40 .When considering the shape of the mountainous surface, the TCCDIF can be described by Formula 13.In parallel to SVF, the adjacent terrain reflections received by mountainous surfaces originate from the portion of the hemisphere obstructed by terrain.Therefore, in estimating reflected radiation from mountainous terrain, the complement of SVF is often utilized as a parameter to quantify the radiation characteristics of this segment 20,46 .This definition is also applied to TCCREF, which is described by Formula 14.
Where, Θ represents the mountain shading factor, which is 0 when the pixel is in the shadow and 1 when it is not shaded.
Following the methodology outlined above, we initially obtained SWDR TCCs at a fine 1 arc-second spatial resolution, which corresponds to the resolution of the DEM.Subsequently, we conducted spatial upscaling on these high-resolution results for two major reasons.Firstly, when compared to coarser spatial scales, TCCs at the 1 arc-second spatial resolution exhibit a heightened sensitivity to potential data production errors 47 .Enhancing the spatial scale of these TCCs serves to mitigate the impact of errors on the terrain correction results.Secondly, many widely used SWDR products, such as MODIS, Himawari-8, GLASS, and others, feature relatively coarse spatial resolutions 5,6,10,14,43,48,49 .Adapting the TCCs products to match these coarser spatial scales offers the advantage of facilitating the direct applicability of the data.
Data accuracy validation based on the three-dimensional radiative transfer model.The evaluation of SWDR accuracy faces notable challenges in mountainous regions.Firstly, there is a lack of observations from stations located on mountainous slopes.Secondly, the spatial distribution of SWDR in mountainous areas is heavily influenced by terrain variations, resulting in a high degree of spatial heterogeneity.Consequently, accurately assessing the accuracy of surface-scale data using point-scale observations is a formidable task 50 .Thirdly, terrain-corrected SWDR outcomes are not only influenced by TCCs but are also significantly reliant on the accuracy of the input SWDR data under the flat surface assumption.
In this context, the challenge lies in establishing a robust methodology for assessing the accuracy of TCCs.Fortunately, the three-dimensional radiative transfer model provides a valuable bridge to furnish a theoretical reference, enabling the evaluation of the accuracy of TCCs.LESS (large-scale remote sensing data and image simulation framework) is a ray-tracing based three-dimensional radiative transfer model capable of achieving rapid, efficient, and accurate simulations of radiative transfer over large regions 51,52 .Within LESS, we employ various scenarios representing different solar incidence angles and surface states to simulate multiple sets of SWDR received by the surface (Table 2).These simulated results are then compared to the correction outcomes achieved through TCCs.It is worth noting that both this study and LESS ignored the anisotropy of diffuse radiation.
By combining the SWDR acquired through the flat surface setting in LESS with TCCs dataset, the terrain-corrected SWDR for the five test areas can be readily computed, as follows:
SWDR SDR D IF REF
Where, E SWDR , E SDR , E DIF , and E REF represent the SWDR, solar direct radiation, diffuse radiation, and reflected radiation from adjacent pixels, which are actually radiation flux density received by the mountainous surface.E SDR H denotes the direct radiation received by a horizontal surface and is equivalent to the Incident solar direct radiation multiplied by the cosine of the SZA.E DIF H represents the diffuse radiation received by the horizontal surface and is equivalent to Incident diffuse radiation.ρ stands for the average surface albedo of the target pixel and its adjacent pixels.In the LESS simulation, the ground surface is modelled as Lambertian with a consistent reflectivity, and thus, ρ aligns with the surface albedo.
Statistical metrics.
We have chosen three statistical metrics to assess the effectiveness of the dataset for SWDR terrain correction: coefficient of determination R 2 , relative root mean square error (rRMSE), and relative bias (rbias).
Coefficient of determination (R 2 ) Relative root-mean-square error (rRMSE) Relative bias (rbias) Where, W i is the SWDR simulated using the LESS model under different mountain surface conditions; P i denote the result of Eqs.15-18; = … i N 1, 2, 3 , (where N is the total number of sample points selected in the study region); and W and P are the average of W i and P i , respectively.R 2 is used to evaluate the closeness of the corre- sponding sample points.The rRMSE is often used to measure the extent of the average error.The rbias indicates the systematic error between two models, which is usually described by overestimation and underestimation.
Data Records
Leveraging SRTM data, we have devised a multi-spatial scale terrain correction parameterization product specifically tailored for the Pan-Third Pole region, encompassing spatial resolutions of 3 arc-seconds, 15 arc-seconds, 30 arc-seconds, 1 arc-minute, and 2.5 arc-minutes.The Pan-Third Pole region shortwave downward radiation topography correction coefficient data set in this study is hosted at the National Tibetan Plateau/Third Pole Environment Data Center (https://doi.org/10.11888/Atmos.tpdc.300784) 53.There are three sets of files: (a) TCCSDR, (b) TCCDIF, and (c) TCCREF.The terrain correction coefficient data is stored in geotiff format with file names following the pattern "TCCSDR_lon_lat_LON_LAT_SZA_SAA_rr.tif", "TCCDIF_lon_lat_LON_ LAT_rr.tif", and "TCCREF_lon_lat_LON_LAT_rr.tif".Among them, lon, lat, LON, LAT, SZA, SAA and rr mean minimum longitude value of image range (°), minimum latitude value of image range (°), maximum longitude value of image range (°), maximum latitude value of image range (°), Solar Zenith Angle (°), Solar Azimuth Angle (°), and rr: spatial resolution, respectively (Table 3).
For example, a file named "TCCSDR_60_20_65_25_10_100_3 s.tif " represents the geotiff file that describes the TCCs data of solar direct radiation in the longitude range of 60-65°E and latitude range of 20-25°N when the SZA is 10° and the SAA is 100°.The TCCs can be calculated as = .× TCCs 0 001 DN, where DN is the digital quantized value.the real TCCs needs to be multiplied by the quantized value in the file by a coefficient of 0.001.
And Fig. 5 shows the topography conditions and spatial distribution of TCCs in some areas used in the accuracy verification process of this article.
Technical Validation
Validate the terrain correction results of solar direct radiation using TCCSDR dataset.To assess the correction results of TCCSDR for solar direct radiation, especially in applications related to vegetation biological processes and solar energy utilization, we conducted a validation by configuring LESS with a surface albedo of 0 as a control.The topographic correction results of TCCSDR under various spatial resolutions and SZAs are depicted in Fig. 6.The R 2 values for all TCCSDR terrain correction outcomes exhibit a high level of agreement with the LESS simulation results, each exceeding 0.96.As the spatial resolution increases, the rRMSE consistently decreases.Notably, at 3 arc-seconds spatial resolution, the R 2 is 0.982, with rRMSE at 8.4% and rbias at −1.0%. Figure 7 shows the SWDR results in mountainous areas simulated by LESS and corrected using TCCs at a spatial resolution of 3 arc-seconds.Meanwhile, at 15 arc-seconds spatial resolution, R 2 stands at 0.996, with rRMSE at 3.3% and rbias at −0.1%.Further improvement is evident at 30 arc-seconds spatial resolution, where R 2 reaches 0.997, with rRMSE at 2.9% and rbias at 0.2%.At 1 arc-minute spatial resolution, the R 2 is 0.997, with rRMSE at 2.6% and rbias at 0.5%.Lastly, at 2.5 arc-minutes spatial resolution, R 2 registers at 0.998, accompanied by rRMSE Table 3. Variable description in data naming.at 2.4% and rbias at 0.8%.Consequently, TCCSDR at varying spatial resolutions demonstrates a reliably high level of accuracy, effectively catering to the diverse data requirements for terrain correction of direct solar radiation.
Validate the terrain correction results of diffuse radiation using TCCDiF dataset.Similar to the validation for solar direct radiation, we evaluated the topographic correction results of TCCDIF for diffuse Fig. 5 The spatial distribution maps of the TCCs and topographic features in the N28E096 region (consistent with region C in Fig. 1 and Table 1).The map is divided into several components: A1 represents the DEM, while A2 and A3 depict slope and aspect, respectively.B1-B3 show the TCCSDR spatial distribution when SZA is 20° and SAA is 90°, with spatial resolutions of 3s, 15 s and 30 s. C1-C3 and D1-D3 are consistent with the B1-B3, but the SZA are 45° and 70°, respectively; E1-E3 and F1-F3 depict the spatial distribution of TCCDIF and TCCREF, respectively.radiation by setting the albedo to 0. TCCDIF effectively performs topographic correction for diffuse radiation across five spatial resolutions, with accuracy consistently improving as the spatial resolution coarsens (Fig. 8).At a 3 arc-second spatial resolution, the results yield an R 2 of 0.940, a rRMSE of 3.2%, and a rbias of −2.1%. Figure 8 also shows the SWDR results in mountainous areas simulated by LESS and corrected using TCCs at a spatial resolution of 3 arc-seconds.Progressing to a 15 arc-second spatial resolution, the R 2 increases to 0.959, with rRMSE at 2.3% and rbias at −1.15%.At a 30 arc-second spatial resolution, the R 2 reaches 0.959, accompanied by rRMSE at 2.0% and rbias at −1.2%.For a 1 arc-minute spatial resolution, the R 2 stands at 0.959, with rRMSE at 1.7% and rbias at −1.0%.Lastly, at a 2.5 arc-minutes spatial resolution, the R 2 reaches 0.965, with rRMSE at 1.2% and rbias at −0.8%.Consequently, TCCDIF efficiently converts diffuse radiation assumed on a flat surface into the actual results received on the mountainous surface, demonstrating effective terrain correction accuracy.
Validate the terrain correction results of SWDR under clear sky conditions using TCCs dataset.The SWDR received by the surface under clear sky conditions primarily comprises solar direct radiation and reflected radiation from adjacent terrain.Consequently, in this segment, we set the surface albedo in the LESS to 0.2 (representing grass) and 0.8 (representing snow).Notably, as sky diffuse radiation contributes insignificantly to the radiation received by the surface under clear sky conditions, we set the sky diffuse radiation to 0. At a spatial resolution of 3 arc-seconds, for low surface albedo (grass), the rbias is 3.4%, the rRMSE is 20.9%, and the R 2 is 0.883.For high surface albedo (snow), the rbias is 3.9%, the rRMSE is 23.0%, and the R 2 is 0845.Coarsening the spatial resolution leads to a slight increase in rbias, a marginal decrease in rRMSE, and an increase in R 2 .When the spatial resolution is further coarsened to 2.5 arc-minutes, the terrain correction results for SWDR using TCCs show an rbias of 5.1%, a RMSE of 7.8%, and a R 2 of 0.989 for low surface albedo representing grass.For high surface albedo representing snow, the rbias is 4.9%, the rRMSE is 8.3%, and the R 2 is 0.979.
The outcomes of the topographic correction for SWDR using TCCs under different SZAs reveal consistent validation results for SZAs of 20° and 45°, both demonstrating excellent accuracy.However, the accuracy diminishes as the SZA increases to 70°.When the spatial resolution is 2.5 minutes in the grass scenario, with a SZA of 20°, the rbias is 1.4%, the rRMSE is 2.1%, and the R² is 0.943 (Fig. 9).For an SZA of 45°, the rbias is 1.9%, the rRMSE is 3.0%, and the R² is 0.972.When the SZA is 70°, the rbias increases to 22.1%, the rRMSE to 25.1%, and the R² decreases to 0.9.In the snow scenario, at the same spatial resolution of 2.5 minutes, when the SZA is 20°, the rbias is 1.2%, the rRMSE is 3.6%, and the R² is 0.687.For an SZA of 45 degrees, the rbias is 1.6%, the rRMSE is 4.3%, and the R² is 0.903.At an SZA of 70 degrees, the rbias is 21.7%, the rRMSE is 24.8%, and the R² is 0.866 (Fig. 10).
In comparison with the results of solar direct radiation, the accuracy of SWDR terrain correction results under clear sky conditions has experienced a decline.This decline in accuracy is attributed to the characterization method of reflected radiation used in this article is still controversial.Chu et al. 54 argue that, at the regional average scale, the complementary value of the sky view factor effectively depicts terrain reflection and aligns with the ray tracing model.Conversely, Shi et al. 27 point out that this method introduces additional errors and fails to provide a precise estimate of the contribution of reflections from adjacent terrain.In spite of the ongoing debate, the method used in this study for terrain correction of terrain reflection is retained due to its simplicity and parametric usability, especially when compared to other methods for calculating adjacent terrain reflection.Users can decide whether to use this method to calculate SWDR terrain reflection according to their own needs.Validate the terrain correction results of SWDR under cloudy sky condition using TCCs dataset.In contrast to the clear sky condition, under cloudy conditions, solar direct radiation struggles to reach the surface directly due to cloud cover under cloudy-sky conditions.The SWDR received by the surface mainly comprises diffuse radiation from the sky and reflected radiation from the adjacent surface.Therefore, when simulating SWDR received by the surface in cloudy skies using LESS, the solar direct radiation is set to 0. The SWDR received by the surface under surface reflectivity representing grass and snow is then simulated.The results of terrain correction using TCCs under cloudy conditions demonstrate consistent performance for both grass and snow.
It is worth noting that, under cloud-sky conditions, the influence of SWDR in mountainous regions manifests in three distinct parts.Firstly, the presence of mountain occlusion, epitomized by the V d , impedes the reception of diffuse radiation within the hemispheric space by the mountainous terrain.Consequently, notwithstanding a diffuse radiation of 1000 W/m 2 from the sky, regions other than open expanses such as mountain peaks and plains receive scattered radiation lower than 1000 W/m 2 .Secondly, the rugged and intricate nature of mountainous terrain contributes to the phenomenon.Although the total radiation flux received by a pixel remains constant, the actual surface area of mountainous terrain exceeds that of flat surfaces, thereby further weaken the unit area density of radiant flux (W/m 2 ).This effect is encapsulated by the area ratio factor (t) in the Eq. 13.Thirdly, mountainous surfaces receive reflected radiation from adjacent surfaces, augmenting the SWDR received.In synthesis, both the first and second parts attenuate the SWDR received by mountainous surfaces.While the third part increases the SWDR received, it is constricted by the preceding two factors.Hence, despite the robust reflection flux on snow-cover surface observed, the radiation flux density received on the surface of mountainous regions remains inferior to that on flat terrain.
It is essential to note that our validation focused on the terrain correction effect of TCCs under ideal clear and cloudy conditions.Real atmospheric conditions often fall somewhere between these two extremes.Although we haven't explicitly verified the accuracy of terrain correction for SWDR composed of direct and diffuse radiation in varying proportions, it can be inferred that its accuracy is a proportional weighting of the clear sky and cloudy sky condition accuracy results discussed in this article.
As indicated by the validation results outlined above, the correction outcomes achieved with our TCCs dataset exhibit a remarkable alignment with the results obtained through ray tracing-based three-dimensional radiation transfer simulations.This implies that our product can significantly assist users in efficiently and accurately implementing SWDR terrain correction in the Pan-Third Pole region.However, it's important to note that in our current study, SZA and SAA are examined at discrete intervals of 5°.In real applications, these angles are continuous rather than discrete.Therefore, when utilizing our product, users still need to interpolate the TCCs values based on the actual SZA and SAA (e.g., the nearest neighbour method).While this interpolation process may introduce some error into the topographic correction of instantaneous SWDR, upscaling the instantaneous results to a daily scale can effectively mitigate its impact.In our forthcoming research endeavours, we are committed to refining the algorithm and developing data products with smaller SZA and SAA intervals.
Fig. 1
Fig. 1 The average slope distribution of the Pan-Third Pole region and five 1°x1° sample areas.
Fig. 4
Fig. 4 Surface area calculation based on irregular triangles.
for solar direct radiation TCCDIF Terrain correction coefficient for diffused radiation TCCREF Terrain correction coefficient for adjacent surface reflected radiation lon Minimum longitude value of image range (°) LON Maximum longitude value of image range (°) lat Minimum latitude value of image range (°) LAT maximum latitude value of image range (
Fig. 6
Fig. 6 Validation of the terrain-corrected solar direct radiation by the TCCSDR in multiple spatial scales/SZAs based on LESS.
Fig. 7
Fig.7 The spatial distribution of solar direct radiation received by the N28E096 region when the SAA is 90°, and the spatial resolution is 3 arc-seconds.The SZAs from left to right are 20°, 45° and 70° respectively.The first line is the result of LESS, and the second line is the result of TCCs.
Fig. 8
Fig.8The first row is validation of the terrain-corrected diffuse radiation by the TCCDIF in multiple spatial scales based on LESS; The second row is the spatial distribution of SWDR received by the N28E096 region simulated by LESS, with a spatial resolution of 3 arc-seconds.The third row is the result of TCCDIF.
Fig. 9
Fig. 9 Validation of the terrain-corrected solar direct radiation by the TCCSDR in multiple spatial scales/SZAs based on LESS, in the grass scenario.
Fig. 10
Fig. 10 Validation of the terrain-corrected solar direct radiation by the TCCSDR in multiple spatial scales/ SZAs based on LESS, in the snow scenario.
Fig. 11
Fig. 11 Validation of the terrain-corrected SWDR by the TCCs in multiple spatial scales based on LESS, under cloudy sky condition.
Table 2 .
Input conditions for LESS. | 2024-07-18T06:17:49.445Z | 2024-07-16T00:00:00.000 | {
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3938611 | pes2o/s2orc | v3-fos-license | Genetic Engineering of Crypthecodinium cohnii to Increase Growth and Lipid Accumulation
In this study, we evaluated suitable selected markers and optimized transformation protocols to develop a new genetic transformation methodology for DHA-producing Crypthecodinium cohnii. Additionally, ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), potentially involved in CO2 fixation under autotrophic conditions, was selected as the target for construction of a gene knockdown mutant. Our results show that the constructs were successfully inserted into the C. cohnii chromosome by homologous recombination. Comparative analysis showed that deletion of the RuBisCO gene promoted cell growth and increased the lipid content of C. cohnii under heterotrophic conditions compared with those of the wild-type. The liquid chromatography-mass spectrometry (LC-MS) based metabolomic analysis showed that the metabolites involved in energy metabolism were upregulated, suggesting that the deletion of the RuBisCO gene may contribute to the re-direction of more carbon or energy toward growth and lipid accumulation under heterotrophic conditions.
INTRODUCTION
The interest in the omega-3 family of long-chain polyunsaturated fatty acids (PUFAs) has increased greatly because these compounds exert recognized beneficial effects on human health (Hornstra, 2000;. Among them, docosahexaenoic acid (DHA) is an important structure component of neural and retinal tissues (Agostoni et al., 1995;Weete et al., 1997). DHA is also a key fatty acid component in breast milk, and is necessary for brain development in infants (Sijtsma and de Swaaf, 2004;Kuratko and Salem, 2013). DHA is widely used in various infant food products (Barclay et al., 1994). The inherent defects of traditional sources of DHA, which is extracted from deep-sea fish oil, limits its application in infant foods (Carlson, 1996). It is therefore necessary to develop alternative sources and technologies for DHA production.
The heterotrophic non-photosynthetic dinoflagellate microalga Crypthecodinium cohnii accumulates lipids with a high fraction of DHA, and is widely used in industrial fermentation for algal oil and DHA production (Harrington and Holz, 1968;Bell and Henderson, 1990;Jiang et al., 1999;Ratledge, 2004).Under optimized cultivation conditions, C. cohnii accumulates less than 1% of the other type of PUFAs (de Swaaf et al., 1999;; this shows remarkable advantages for the downstream DHA purification process. Recently, much effort has been placed into improving DHA accumulation in C. cohnii. For example, optimization of the fermentation parameters in fed-bath experiments led to a production of 109 g/L dry biomass, 61 g/L lipid, and a 19 g/L DHA in C. cohnii (de Swaaf et al., 1999Sijtsma and de Swaaf, 2004). In addition, chemical triggers such as butylated hydroxyanisol (BHA) (Sui et al., 2014) and ethanolamine (Li et al., 2015) were applied to directly stimulate lipid accumulation in C. cohnii. However, no study thus far has reported on using genetic engineering on DHA-producing C. cohnii. Several studies have successfully applied genetic engineering to improve lipid content in various microalgae (Radakovits et al., 2011;Hamilton et al., 2014;Iwai et al., 2014;Avidan et al., 2015). Thus, it is necessary to develop relevant methodologies for genetic engineering of C. cohnii with highefficiency production of DHA. Recently, transformation systems have been developed for several other dinoflagellate species, such as Amphidinium and Symbiodinium (Te and Miller, 1998); however, no transformation system has yet been established for C. cohnii.
High-efficiency production of target products requires re-balancing of energy and carbon flux in cells (Komatsu et al., 2010). For example, lipid production is maximized in Yarrowia lipolytica by engineering a cytosolic redox metabolism to increase the supply of NADPH (Qiao et al., 2017). Limonene production is enhanced by portioning a greater carbon flux to 2-C-methyl-D-erythritol 4-phosphate pathway . C. cohnii can be derived from a photosynthetic ancestor and harbors a reduced plastid (Sanchez-Puerta et al., 2007). The ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in non-photosynthetic C. cohnii, cultivated under heterotrophic conditions, is still transcribed (Sanchez-Puerta et al., 2007). Typically, RuBisCO is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted to energy-rich molecules such as glucose (Dhingra et al., 2004). In addition, RuBisCO may act as an oxygenase in photorespiration, and is involved in glycine and serine biosynthesis (Leegood, 2007). The energy source ATP and reducing equivalent NADPH are consumed in the two aforementioned reactions. To date, the specific roles of RuBisCO in non-photosynthetic C. cohnii are not clearly elucidated. However, with respect to the metabolic economy of a cell, pathways involved in CO 2 fixation and photorespiration may not be essential under heterotrophic conditions. For example, Rhodobacter sphaeroides and Rhodopseudomonas palustris, the RuBisCO gene deletion mutants, can save more energy and reductants under chemoheterotrophic conditions (Laguna et al., 2011). Therefore, we hypothesized that in C. cohnii, the deletion of these high abundance but nonessential genes, such as RuBisCO, may optimize the carbon flux and redirect the energy flux (ATP and NADPH) that are consumed in the Calvin-Benson-Bassham (CBB) cycle of cell growth and lipid accumulation under heterotrophic conditions. To engineer C. cohnii for high-efficiency lipid and DHA production, we first developed a genetic transformation system for C. cohnii, and then applied for the construction of a RuBisCO-deleted strain. Our results show that deletion of the RuBisCO gene promoted cell growth and increased lipid content of C. cohnii under heterotrophic growth conditions compared with those of the wild-type. To explore the possible mechanism, a liquid chromatography-mass spectrometry (LC-MS) based metabolomics analysis was used to compare a RuBisCO-deleted and wild-type C. cohnii; this analysis provided new insights into the metabolic changes associated with increased growth and lipid accumulation in C. cohnii. The obtained information will be useful for engineering high-efficiency lipid and DHA-producing strains in the future.
Algae Strains and Cultivation
Crypthecodinium cohnii ATCC 30556 was obtained from American Type Culture Collection (ATCC) and cultivated in a basal medium according to Sui et al. (2014). The seed cultures were cultivated in 50 mL of basal medium (pH 6.5) containing 9.0 g/L glucose, 2.0 g/L yeast extract (OXOID, Basingstoke, United Kingdom), and 25.0 g/L sea salt (Sigma-Aldrich, St. Louis, MO, United States), in 250-mL Erlenmeyer flasks at 25 • C, shaken at 180 rpm.
Preparation of C. cohnii Competent Cells
Crypthecodinium cohnii cells (10 mL) in exponential phase were harvested by centrifugation at 3500 rpm (Eppendorf 5430R, Hamburg, Germany) at 4 • C for 5 min. The collected cells were re-suspended in 1 mL of 25 mM DTT and pretreated at 30 • C for 15 min. After the pretreatment, cells were harvested again by centrifugation at 3500 rpm (Eppendorf 5430R, Hamburg, Germany) at 4 • C for 5 min, and washed three times with 10 mL of 1 M sorbitol. Finally, cells were re-suspended in 1 mL of 1 M sorbitol.
Electroporation With FITC-Dextran as Fluorescence Marker
The final mixture of competent cells (200 µL), 1 µL sperm DNA (100 mg/mL), and 1 µg FITC-Dextran (70 kDa) were transferred into a pre-chilled 2 mm gap electroporation cuvette (Bio-Rad, Hercules, CA, United States), ice bathed for 5 min, after which different electric fields were applied via electroporation system (Gene Pulser Xcell; Bio-Rad, Hercules, CA, United States). After the electric shock was applied, the cells in the cuvette were washed twice with basal medium, re-suspended in 2 mL basal medium, and treated with 1 mg/mL trypsin at 37 • C for 15 min (Richard et al., 2003).
Analysis of Viability and Fluorescence
Fluorescence intensity in the transformed cells was measured by a spectrophotometer (F-2700EL, HITACHI, Chiyoda, Japan).
The excitation wavelength for FITC-Dextran was 490 nm; the highest peak at the emission wavelength for FITC-Dextran (520 nm) was used to calculate the transformation efficiency. The transformed cells were also observed under a fluorescence microscope (OLYMPUS, BX43, Shinjuku, Japan).
Antibiotics Sensitivity Assessment of C. cohnii
Crypthecodinium cohnii cells (50 µL) of the exponential phase were selected on plates containing different concentrations of hygromycin (10-50 mg/L) or bleomycin (10-200 mg/L), and cultivated at 25 • C for up to 2 weeks.
Constructs Used for Transformation
Phusion high-fidelity DNA polymerase (Thermo-Fisher, Waltham, MA, United States) was used in the PCR reactions. A fusion PCR-based method was employed for the construction of gene knockout fragments (Wang et al., 2002). For the target genes selected, three sets of primers were designed to amplify a linear DNA fragment containing the hygromycin or bleomycin resistance gene with two flanking arms of DNA upstream and downstream of the targeted gene. The linear fused PCR amplicon was used directly for transformation into C. cohnii by electroporation. Hygromycin and bleomycin resistance genes were amplified from pCAMBIA1301 (Hajdukiewicz et al., 1994) and pSP124s (Lumbreras et al., 1998), respectively. PCR primers used for mutant construction are listed in Supplementary Table S1.
Transformation of C. cohnii
A 200-µL mixture of competent cells (prepared as mentioned in section "Electroporation With FITC-Dextran as Fluorescence Marker"), 1 µL sperm DNA (100 mg/mL), and 1∼2 µg DNA fragment were transferred into a 2-mm pre-chilled electroporation cuvette and ice bathed for 5 min. Gene Pulser Xcell (Bio-Rad, Hercules, CA, United States) was used for electroporation, and the electroporator was adjusted to 2000 V field strength and 50 µF capacitance. After electroporation, the cells were recovered in 2 mL of basal medium, and cultivated for 48 h at 25 • C while shaking at 180 rpm. Then, 50 µL of cell suspension were selected on basal medium plates containing antibiotics of suitable concentration, as described in section "Antibiotics Sensitivity Assessment of C. cohnii."
Mutant Screening and Analysis
Hygromycin or bleomycin-resistant transformants on the plates were streaked onto fresh basal medium plates supplemented with hygromycin or bleomycin, and passaged several times. Mutants were confirmed by PCR and sequencing analysis. TIANamp Bacterial DNA Kit (TianGen, Beijing, China) was used to extract chromosomal DNA of wild-type and mutant C. cohnii. PCR primers used for validating mutants are listed in Supplementary Table S1.
Comparative Growth Analysis and LC-MS Targeted Metabolomics
Cell density of wild-type and mutant C. cohnii was determined by a UV-1750 spectrophotometer (Shimadzu, Kyoto, Japan) at OD 490 . For LC-MS metabolomic analysis, all reagents, including standard metabolites, were obtained from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, United States). Cells were harvested by centrifugation at 8000 × g for 8 min at 25 • C (Eppendorf 5430R, Hamburg, Germany). Metabolite extraction and LC-MS analysis were performed according to a previously published protocol (Li et al., 2015). Metabolomic data were normalized by interior control and cell number, and then submitted to principal component analysis (PCA) using SIMCA-P 11.5.
Semi-quantitative and Quantitative RT-PCR Analyses
(i) Semi-quantitative RT-PCR analysis: total RNA was extracted from cells with Trizol reagent (Invitrogen, Camarillo, CA, United States) according to the manufacturer's instructions. RT-PCR was conducted using cDNAs generated from 1 µg of total RNA with a RevertAid First Strand cDNA Synthesis Kit (Thermo-Fisher, Waltham, MA, United States). The RT-PCR exponential phase was determined using 22-30 cycles, to allow for semiquantitative comparisons of cDNA developed using identical reactions with Taq polymerase (Tiangen, Beijing, China); the method is described in Song et al. (2016). The housekeeping gene 18S rDNA (GenBank accession no. FJ821501.1) of C. cohnii was used as internal reference.
(ii) Quantitative RT-PCR analysis was conducted according to the method described previously (Song et al., 2016). Briefly, a StepOne Plus Real-Time PCR (Applied Biosystems, Foster City, CA, United States) was used to conduct the analysis. Cell pellets were re-suspended in Trizol reagent (Invitrogen, Camarillo, CA, United States) and mixed thoroughly by vortexing. Total RNA extraction was achieved using a miRNeasy Mini Kit (Qiagen, Valencia, CA, United States). Contaminating DNA in RNA samples was removed with DNase I according to instructions in the miRNeasy Mini Manual (Qiagen, Valencia, CA, United States). The RNA quality and quantity were determined using NanoDrop 2000 (Thermo-Fisher, Waltham, MA, United States) and subjected to cDNA synthesis. Then, 2 − C T was used to estimate the relative abundance of different mRNA molecules: the higher the C T value, the less abundant is the corresponding mRNA (Livak and Schmittgen, 2001).
Enzyme Activity Assay and Western Blotting
RuBisCO enzyme activity was determined using the 14 C isotopebased method described by Lorimer et al. (1977). Afterward, protein extraction was conducted according to Lilley et al. (2010). The assay was performed in a 460-µL final volume containing 50 mM Tris (adjusted to pH 8.1 with HCl at 20 • C), 10 mM EDTA, 20 mM MgCl 2 , 10 mM NaH 14 CO 3 (Sigma-Aldrich, St. Louis, MO, United States), and 20 µL crude extracts. The reaction was started by adding 20 µL of 10 mM RuBP (Sigma-Aldrich, St. Louis, MO, United States). The reaction was stopped after 1 min by adding 200 µL of 2 M HCl. The contents of the vial were quantitatively transferred to a scintillation vial, dried, and quantified per a previously described protocol (Sudhani et al., 2015).
For western blotting assay, total protein extraction was conducted according to Lilley et al. (2010). Then, 10 µl of loading buffer (Tiangen, Beijing, China) was added to 20 µl of crude extracts, and the mixture was heated for 10 min at 100 • C. The samples were loaded onto a precast polyacrylamide gel. After SDS-PAGE, the separated proteins were electroblotted onto a polyvinylidene fluoride (PVDF) membrane and probed with an affinity-purified antibody against the large subunit of RuBisCO (AS03 037, Agrisera, Vännäs, Sweden). After incubating with a secondary antibody [horseradish peroxidase-conjugated goat anti-rabbit IgG; AS 09602, Agrisera, Vännäs, Sweden], ECL Prime (GE, Amersham, United Kingdom) was used for detection. The detection analysis was conducted using a CCD camera according to the manufacture's instructions.
Lipid Extraction and Analysis
Crypthecodinium cohnii cells were cultivated in basal medium with 21 g/L glucose, harvested at 84 h by centrifugation (3500 × g) (Eppendorf 5430R, Hamburg, Germany), and freezedried to generate a lyophilized algal powder. Lipid extraction and analysis were conducted based on the method described by Li et al. (2015).
Statistical Analysis
All experiments were conducted with at least three biological replicates to ensure reproducibility. Statistical analyses were performed using a one-way analysis of variance (ANOVA) with subsequent post hoc multiple-comparison LSD tests, which were calculated using SPSS Statistics software. Statistically significant values were defined as P < 0.05.
Establishing a Transformation Protocol
Crypthecodinium cohnii, a heterotrophic dinoflagellate alga, is an important microalgal species for industrial fermentation of algal oil and DHA production. However, because of the lack of genetic transformation systems, no genetic engineering work has been conducted on this valuable species. This has restricted the efforts in strain improvement and deciphering the mechanism of DHA accumulation in C. cohnii. Macromolecules are successfully transferred into protoplasts or spheroplasts of C. cohnii by liposomal-mediated transformation (Kwok et al., 2007). However, preparation of protoplasts or spheroplasts can be challenging and time consuming. Electroporation is a powerful method for genetic engineering of microalgae (Sun et al., 2005;Weeks, 2011). To establish an efficient transformation system for C. cohnii, we first selected the macromolecule FITC-Dextran (70 kDa) as a fluorescent marker to evaluate the electroporation parameters for C. cohnii. As shown in Figure 1A, the fluorescence values of the transformed cells (1.6 kV, 200 , 50 µF) were slightly increased when compared with those of the control cells, suggesting that the macromolecule FITC-Dextran was delivered into C. cohnii cells by electroporation. The results of examination via fluorescence microscopy ( Figure 1B) confirmed that the increase of fluorescence values was due to the delivery of FITC-Dextran into C. cohnii cells. To improve transformation efficiency, we further optimized the electroporation parameters by applying voltages ranging from 1.6 to 2.4 kV, because higher electric shocks can lead to more uptake of extracellular molecules by the cells (Jeon et al., 2013). When voltage was adjusted to 2.0 kV, we observed the maximum intensity of fluorescence; however, further increase of voltage to 2.2 or 2.4 kV led to a decrease of fluorescence intensity in the cells (Figure 1A). According to our optimization analysis using FITC-Dextran, an electroporation protocol with parameters of 2.0 kV, 200 , and 50 µF was determined for the next procedure using C. cohnii cells.
Transformation of C. cohnii
In order to determine whether DNA constructs could also be transferred and integrated by homologous recombination (HR) into C. cohnii chromosomal DNA, we selected gene encoding 18S rDNA as a suitable site for HR (Cheng et al., 2010;Yan et al., 2013). 18S rDNA exists in multiple copies in the chromosomes and is located at transcriptionally active regions in organisms. This renders 18S rDNA more suitable for integration of foreign DNA than other sites. In addition, insertion inactivation of at least some copies of 18S rDNA is not lethal (Pisabarro et al., 1998). However, it is necessary to identify appropriate selectable makers for transformation. As shown in Supplementary Figure S1, when the concentrations of hygromycin and bleomycin were increased to 40 mg/L and 200 mg/L, respectively, no growth was observed on the plates containing the antibiotics; this suggests that 40 mg/L hygromycin and 200 mg/L bleomycin can be suitable concentrations used for selection, respectively. The transformation construct C1 carried the hygR resistant gene, which conferred resistance to hygromycin as a selection marker (Supplementary Figure S2A). For transformation, 2 µg of construct C1 DNA was used, which gave three possible positive clones (i.e., strain 3, 5, 7). After streaking the clones onto fresh basal medium in a plate containing 40 mg/L hygromycin, growth was observed only for strain 5 (Figure 2A). To further validate whether the selected marker gene hygR was successfully integrated into the genome, we conducted PCR with primer sets (P-F and Hyg-R2) to detect the hygR selected marker gene using chromosomal DNA as template ( Figure 2B). The amplicon was purified, sequenced, and confirmed. The results demonstrated the integration of hygR gene into the C. cohnii genome (data not shown), suggesting a successful establishment of the C. cohnii transformation protocol. The detailed transformation protocol is provided in Supplementary Figure S3.
Construction of RuBisCO-Deletion Mutants
The RuBisCO gene in non-photosynthetic C. cohnii, cultivated under heterotrophic conditions, is still transcribed (Sanchez-Puerta et al., 2007). Because the reactions involving RuBisCO consume energy and reducing equivalents (Dhingra et al., 2004;Leegood, 2007), we next aimed to redirect energy and reducing equivalents toward cell growth and lipid biosynthesis; for this, we constructed the RuBisCO-deleted mutants using the abovementioned transformation protocol for C. cohnii. Until now, the information about the structure and sequence of C. cohnii RuBisCO gene was lacking. However, studies on the dinoflagellate Prorocentrum minimum showed that over 10 transcribed units of the RuBisCO gene are present in the genome, and each transcribed unit contains four tandem copies of the RuBisCO coding region (1.46 kb each) interspersed by a 63-bp spacer. It was also estimated by real-time PCR analysis that each P. minimum genome harbors 148 ± 16 coding regions (Zhang and Lin, 2003). The sequence analysis of the coding regions revealed that the nucleotide sequences vary by approximately 1.0-9.0% among the coding regions; however, their inferred amino acid sequences are essentially identical (Zhang and Lin, 2003). Accordingly, we hypothesized that extensive gene duplication may also exist for the RuBisCO gene in C. cohnii. The RuBisCO gene has been found in the C. cohnii nuclear genome; this gene shares an evolutionary history with form II RuBisCO genes from peridinin-containing dinoflagellates (Sanchez-Puerta et al., 2007). Based on the sequence of the RuBisCO gene of C. cohnii (GenBank accession no. EB086308.1), we designed PCR primers (Ru-F and Ru-R) for amplification of RuBisCO gene fragments using both C. cohnii cDNA and genomic DNA as template, respectively. Two clear fragments of different sizes, designated as Ru-C and Ru-G, respectively, were obtained by PCR (Supplementary Figure S4). Sequencing and blast analysis showed that the Ru-C fragment matched to the reported C. cohnii RuBisCO gene, while the Ru-G fragment showed no significant similarity (data not shown). While we cannot rule out the possibility that Ru-G was from a different copy of the RuBisCO gene, we performed a phylogenetic analysis of this fragment with several known algal RuBisCO genes, including those from Prorocentrum minimum, Karenia mikimotoi, and Symbiodinium sp. The results showed that both Ru-C and Ru-G were clustered with other RuBisCO genes, suggesting that both could be putative RuBisCO genes in C. cohnii (Supplementary Figure S5). Obvious similarity was also observed when the DNA sequence of Ru-G was aligned against other RuBisCO genes from dinoflagellates and prokaryotes (Supplementary Figure S6). We first targeted the Ru-C fragment for the construction of a single-deletion mutant (construct see Supplementary Figure S2B). Three positive deletion clones (M-1, M-6, and M-7) were confirmed by growth patterns on plates with antibiotics ( Figure 3A, top panel) and PCR analysis using a primer set of P-F and Hyg-R2 ( Figure 3A, bottom panel). Using single-deletion mutants, we then targeted the Ru-G fragment for constructing double-deletion mutants (construct see Supplementary Figure S2C). The double-deletion mutants of both Ru-C and Ru-G were obtained and confirmed by double resistances and PCR analysis using a primer set of Ble-F3 and Ble-R2 ( Figure 3B). In addition, to validate whether the resistance gene was successfully integrated into the genome, a PCR analysis with primers specific to RuBisCO gene and resistance gene yielded the expected amplification products in each of the mutants (i.e., primer sets of Hyg-F3 and Ru-R1, and Ble-F3 and Ru-R2, respectively) (Supplementary Figure S7). To further confirm the knockout of these two RuBisCO genes in C. cohnii, we also performed a RT-PCR analysis of the expression levels of the RuBisCO gene, 14 C isotope-based enzymatic assay of RuBisCO activity, and western blotting analysis of the abundance of the RuBisCO protein in both WT and mutant cells. The semiquantitative RT-PCR results showed that RuBisCO gene RNA expression was clearly decreased in the mutants (Figure 4A), and the RuBisCO enzymatic activity assay showed an approximately 30% decrease of the activity in all mutant cells (Figure 4B). These results suggest that the RuBisCO gene was transcribed and translated in C. cohnii under heterotrophic growth conditions, and the RuBisCO protein retained the specific carboxylation activity. In addition, the abundance of the RuBisCO protein was also clearly decreased in both single-and double-deletion mutants, as shown by western blotting (Figure 4C). Altogether, these data demonstrate that the knockdown of RuBisCO gene was evidenced in C. cohnii.
We found that the RuBisCO gene cannot be fully deleted. Two possible explanations are as follows. Copies of the RuBisCO gene are abundant in dinoflagellates (Zhang and Lin, 2003;Shi et al., 2013); currently, we were not able to delete all the RuBisCO genes due to the lack of the whole-genome sequence of C. cohnii. The mutants constructed were thus only knockdown mutants of the overall expression level of the RuBisCO genes in C. cohnii. It is also possible that RuBisCO is an essential gene at heterotrophic conditions; this notion is supported by the fact that the RuBisCO gene is transcribed and translated in C. cohnii under heterotrophic condition (Sanchez-Puerta et al., 2007). In Ralstonia eutropha H16, two RuBisCO genes in the CBB cycle are actually functional in CO 2 fixation under heterotrophic conditions (Shimizu et al., 2013). Moreover, in Rhodobacter sphaeroides and Rhodopseudomonas palustris, the RuBisCO deletion mutants were lethal under malate-dependent photoheterotrophic conditions. This indicates the essential role of the CBB cycle in maintaining the redox balance in cells for efficient use of the carbon source (Laguna et al., 2011). Taken together, these results suggest that the RuBisCO gene may be essential for growth. In addition, RuBisCO can also function as an oxygenase involved in other metabolic activities such as glycine and serine biosynthesis (Wolfe and dePamphilis, 1998;Sekiguchi et al., 2002). Although the activity of RuBisCO enzyme was decreased in both single-and double-deletion mutants, no significant decrease was observed in the double-deletion mutants when compared with the single-deletion mutants. This may be due to differential regulation, or roles, of the different RuBisCO genes under various growth conditions. For example, expression of a gene encoding a truncated large subunit of RuBisCO is salt-inducible in rice (Zhang et al., 1995), and an organ-specific expressed subunit of RuBisCO can alter the catalytic properties of the RuBisCO holoenzyme in rice (Morita et al., 2014).
Comparative Growth and Lipid Content Analysis
To demonstrate that the knockout of the RuBisCO gene was metabolically advantageous to C. cohnii cells, we conducted a comparative growth analysis of the RuBisCO mutants and wild-type; this analysis was performed on basal media with a glucose concentration ranging from 9 to 27 g/L. As shown in Figure 5, the deletion of the RuBisCO gene in C. cohnii resulted in enhanced cellular growth. Growth increase was more significant at a high glucose concentration of 21 g/L than at the basal concentration of 9 g/L. However, when glucose concentration reached 27 g/L, enhancement of growth in the mutants became less significant ( Figure 5D). Under this condition, although the growth of the mutants was slightly slow at early exponential phase, it caught up and reached higher levels at the stationary phase ( Figure 5C). Growth increases in the RuBisCO-deleted C. cohnii were also confirmed by statistical analysis with p-values less than 0.05; the results showed that at the late growth phase (i.e., stationary phase), the accumulated growth differences were more significant at all the tested concentrations of glucose. We also examined lipid accumulation in the wildtype and in RuBisCO-deleted mutants during the late growth phase, when the most significant increase in cell growth was observed ( Figure 5C). Cells were harvested at 84 h and extracted for total lipids. We observed a significant increase in lipid accumulation, with an average improvement of approximately 10.6% (Figure 6).
Targeted Metabolomic Analysis
We next explored the possible molecular mechanisms contributing to the enhanced growth and lipid accumulation in RuBisCO-deleted mutants. For this, we used LC-MS-based targeted metabolomics to quantify the time-series changes of 24 selected metabolites related to central carbohydrate and energy metabolism in C. cohnii cells. Using the optimized protocol described previously (Sui et al., 2014;Li et al., 2015), we achieved reproducible analysis of 24 selected intracellular metabolites for C. cohnii. For the metabolomic analysis, two single-deletion and two double-deletion RuBisCO mutants, and wild-type C. cohnii, were selected and cultivated for 4 days in a medium with 21 g/L glucose. Cell samples were FIGURE 6 | Lipid accumulation in the RuBisCO-deletion mutants and wild-type. The lipid content in the control was set as 100%, and percentage changes in the mutants are shown. Data represent the mean ± SD of three independent experiments. Asterisks indicate significant differences between the control and mutants, as evaluated by Student's T-test ( * p < 0.05).
collected at 48 and 84 h, which corresponds to exponential and stationary growth phases, respectively. The time-series changes in targeted metabolites were determined using the LC-MS approach (raw metabolomic data are provided in Supplementary Table S2). For each sample, three biological replicates were prepared and collected separately, and each of the biological replicates was analyzed twice by LC-MS.
The quality of the LC-MS metabolomics analysis was confirmed by a principal component analysis (PCA) (Supplementary Figure S8). Moreover, mutant cells were at the farthest distance from the controls at 84 h, as shown in Supplementary Figure S8B; this indicates the most significant metabolic changes in cells at this time point, consistent with their growth patterns ( Figure 5C). To better interpret the qualitative information of the results, we generated heatmaps of the two time points using MultiExperiment Viewer software. Similar methodology has been successfully applied to analyze transcriptomic data previously (Putri et al., 2013), in which the ratio between the abundance of the metabolite under a given condition, and the average abundance of the metabolite in all samples, was calculated for each metabolite. The analysis showed that at 48 h, changes in the metabolites were detectable for single-deletion mutants. However, the fold changes were relatively small. The changes were not obvious for the double-deletion mutants, which was consistent with their growth patterns. At 48 h, most of the detected metabolites were downregulated, except for ADP-Glucose, G6P, ATP, ADP, CoA, NADH, and AMP in the RuBisCO singledeletion mutants. Nearly all metabolites were downregulated in the double-deletion mutants compared with the wild-type (Figure 7A). At 84 h when the most significant differential growth was recorded (Figure 5C), we observed upregulation of most metabolites in both single-and double-deletion mutants; these metabolites included F6P, NADP, NADPH, UDP-Glucose, Ac-CoA, ADP, ATP, RiBP, DHAP, GAP, and Glu ( Figure 7B). Thus, we hypothesized that deletion of the RuBisCO gene in the CBB cycle may disrupt the redox balance in the plastid, thereby enhancing the central carbohydrate and energy metabolism to improve cell growth and lipid accumulation.
These results showed an upregulation of NADH and downregulation of NAD, which increased the ratio of NADH/NAD at 48 h in the single-deletion mutant ( Figure 7A). The high ratio of NADH/NAD provides a reducing environment, beneficial to cell growth (Schwartz et al., 1974). However, at 84 h, the ratio of NADH/NAD in the wild-type and mutants had barely changed, and the content of Ac-CoA, ATP, and NADPH were improved; this indicates that multiple mechanisms may be involved at different growth phases. For example, the metabolite Ac-CoA can induce cell growth and proliferation by promoting the acetylation of histones at growth genes (Cai et al., 2011). Increasing the content of intracellular NADPH by improving the activity of glucose-6-phosphate dehydrogenase also stimulates cell growth (Stanton, 2012). In addition, AKG can behave as an inhibitor of malic enzyme, which is considered a key pathway for the generation of NADPH (Gupta et al., 2013). Therefore, the decreased levels of AKG in the mutants may result in reduced inhibition of the generation of NADPH. The downregulation of AKG was consistent with the results of previous studies (Ratledge, 2002;Sui et al., 2014), showing that a decrease in AKG releases inhibition of NADPH biosynthesis, leading to increased fatty acid biosynthesis (Ratledge, 2002). Moreover, the energy currency ATP was upregulated, which is beneficial for efficient transcription and protein synthesis (Gaal et al., 1997). This is consistent with a study on Escherichia coli, showing that intracellular ATP concentration was found to increase proportionally with the growth rate (Schneider and Gourse, 2004). Moreover, when cells enter the phase in which they accumulate lipids, the pentose phosphate pathway (PPP) is downregulated (Sui et al., 2014;Wase et al., 2014;Li et al., 2015); our analysis consistently showed that R5P was downregulated.
Because our metabolomics analysis indicated the upregulated energy metabolism as one of the possible reasons for enhanced growth in the RuBisCO-deleted mutants, we next validated these results using quantitative RT-PCR. For this, we compared the expression levels of two selected genes, ATP synthase and ribosome coding genes, in wild-type and mutant cells. We hypothesized that if cells can retain a high growth rate, the expression level of ATP synthase and ribosomes must be increased to maintain a high-level supply of ATP and proteins. The results show that both genes were significantly upregulated in the mutants (Supplementary Figure S9); this is consistent with enhanced energy metabolism, as shown by the metabolomics analysis. In the CBB cycle, one molecular CO 2 fixation consumes 3 molecular ATPs and 2 molecular NADPH (Alric et al., 2010). If the RuBisCO gene is deleted, more ATP and NADPH is saved (Laguna et al., 2011). These results support our hypothesis that the deletion or knockdown of the highly abundant, but unnecessary gene, RuBisCO in the CBB cycle may disrupt the redox balance in the plastid. This is beneficial for redirecting the carbon and energy to cell growth and lipid accumulation under conditions of heterotrophic growth. However, the molecular mechanism of how redox unbalancing in the RuBisCO-deleted mutant enhances the central carbohydrate and energy metabolism needs to be further elucidated. FIGURE S5 | Neighbor-joining (NJ)-based phylogenetic analysis of Ru-C (C. cohnii-1), Ru-G (C. cohnii-2), and RuBisCO. This phylogenetic analysis was based on nucleotide sequences, and Clustal X and MEGA4 were utilized to produce the phylogenetic tree. | 2018-03-19T17:21:18.596Z | 2018-03-19T00:00:00.000 | {
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232368447 | pes2o/s2orc | v3-fos-license | Loss of Ftsj1 perturbs codon-specific translation efficiency in the brain and is associated with X-linked intellectual disability
Defective tRNA 2′-O-modification impairs translation efficiency at Phe codon in the brain and induces a neurological dysfunction.
INTRODUCTION
Transfer RNA (tRNA) is central to the decoding of the genetic information into polypeptide during translation. The nucleotides of tRNAs contain more than 100 species of modifications, which are posttranscriptionally introduced by specific enzymes (1,2). tRNA modifications regulate structural integrity, stability, and codonanticodon interaction and thus contribute to translation efficiency (TE) and fidelity. Accumulating evidence has shown that defective modifications can impair optimal protein synthesis in humans and cause a variety of diseases including cancer, type 2 diabetes, and mitochondrial and neurological diseases (3).
Intellectual disability (ID) is a neurological disorder that affects 2 to 3% of individuals worldwide (4). Clinical symptoms of ID include cognitive dysfunction, defined by an intelligence quotient of <70, and disability in adapting to social environments (5). The underlying cause of ID can be both genetically inherited and acquired (5). Defects in genes on the X chromosome are major contributors to ID (6), with X-linked ID (XLID) estimated to comprise 5 to 16% of all male patients with ID (6)(7)(8). To date, a total of 141 X-linked genes have been identified as causal genes for XLID (6). However, despite rapid progress in finding candidate genes, the pathogenic mechanisms underlying XLID have remained largely unknown.
The human FtsJ RNA 2′-O-methyltransferase 1 (FTSJ1) gene is localized at position Xp11.23 on the X chromosome, and pathogenic mutations of FTSJ1 have been associated with development of XLID in Belgian, Japanese, and Chinese families (8)(9)(10)(11)(12). Splice site mutations, nonsense mutations, and microdeletions have been identified in both coding and intron regions of FTSJ1, which results in production of abnormal FTSJ1 transcripts. Patients having a defective FTSJ1 gene manifest moderate to severe intellectual handicap characterized by delayed speech and disabilities in reading and extracting word meaning. Some patients display behavior problems such as aggressive outbursts, anxiety, and panic. Besides these intellectual and behavioral symptoms, these patients show no dysmorphism, indicating that patients having defective FTSJ1 gene develop nonsyndromic XLID.
Transfer RNA methyltransferase 7 (Trm7), the yeast homolog of Ftsj1, catalyzes 2′-O-methylation of cytidine at position 32 (C32) and guanosine at position 34 (G34) of tRNA Phe , C32 and C34 of tRNA Trp , and uridine at position 34 (U34) of tRNA Leu (13). At a cellular level, Trm7-deficient yeast are viable but show growth defects. Expression of human FTSJ1 in Trm7-deficient yeast restores 2′-O-methylation, leading to recovery of cell growth (14). Overexpression of tRNA Phe , but not other Trm7 substrate tRNAs, can rescue the growth defect of Trm7-deficient yeast cells (15). In human-derived cell lines, FTSJ1 appears to modify a broad number of tRNA species, and loss of FTSJ1 induces abnormal translation of a luciferase-based reporter (16,17). Ftsj1-deficient animal models have been generated using Drosophila and mouse (18,19). Drosophila with deficiency of Ftsj1 homologs shows defective 2′-O-methylation at tRNA Phe along with several other tRNAs and exhibits broad phenotypes including life-span reduction and dysfunction of small RNA pathways (18). Recently, a line of Ftsj1 mutant mice was generated using a gene-trapping method (19). Although the level of tRNA 2′-O-methylation in the Ftsj1 gene trapped mice has not been examined, the mutant male mice showed an abnormality in a place-learning paradigm (19). The mutant mice also showed a decrease in bone density and bodyweight (19). Despite these findings, two fundamental questions remain unsolved: (i) Why loss of FTSJ1 causes nonsyndromic ID despite the ubiquitous expression of FTSJ1, and (ii) how loss of functional Ftsj1 leads to the development of XLID.
Here, we report comprehensive analyses of constitutive Ftsj1 knockout (KO) mice and a cell line derived from the patient with FTSJ1-related XLID, using biochemical, molecular, physiological, and behavioral approaches. We found that tRNA Phe , a Ftsj1 substrate tRNA, selectively decreased in the brain. Ftsj1 KO mice showed selective translational perturbation at Phe codons, leading to the abnormal translation of a subset of genes implicated in neuronal function. Furthermore, Ftsj1 KO mice showed morphological and electrophysiological deficits, which were associated with behavioral abnormalities that recapitulate symptoms of FTSJ1-related XLID.
Loss of tRNA 2′-O-methylation in Ftsj1 KO mouse and XLID patient-derived cells
We generated Ftsj1 KO mice (fig. S1A), which developed normally albeit with a slight but significant decrease in body weight compared to wild-type (WT) mice at 4 weeks of age ( fig. S1B). According to the modification database (2), at least 11 species of tRNAs contain 2′-O-methylated nucleotides at position 32 and/or 34 in mammals. To investigate whether Ftsj1 is responsible for 2′-O-methylation of the 11 species of tRNAs, we isolated these tRNAs from the liver of 8-week-old WT and KO mice and examined by mass spectrometry. 2′-O-methylation at position 32 and/or 34 could not be detected for 11 species of tRNA in Ftsj1 KO mice including tRNA Phe , a known Ftsj1 substrate (Fig. 1, A to C, and fig. S1, C and D). tRNA Phe contains a hypermodified guanosine at position 37, the wybutosine (yW) modification in yeast and the hydroxywybutosine (OHyW) modification in human (2). G37 of tRNA Phe was modified to OHyW with 95% efficiency in WT mouse liver, but modification was reduced to 47.9% in KO mouse liver (Fig. 1, B and C). These results are consistent with previous studies showing that 2′-O-methylation at C32 and G34 is required for subsequent hypermodification of tRNA Phe at G37 in eukaryotic cells (14,16,17). In addition, we did not detect 2′-O-methylation at position 32 and/or 34 of all tRNA Leu isoacceptors [tRNA Leu(UAA) , tRNA Leu(CAA) , tRNA Leu(AAG) , tRNA Leu(UAG) , and tRNA Leu(CAG) ], tRNA Trp , tRNA Sec , tRNA Val(AAC) , tRNA Gln(CUG) , and tRNA Arg(ACG) in Ftsj1 KO mice (fig. S1C).
We also examined 2′-O-methylation of tRNAs in an immortal lymphocyte cell line established from a patient with XLID and control cell lines from their parents. This patient has a G-to-A substitution at the splice donor site in intron 8 (c.571+1G>A), which caused a retention of the entire intron 8 in FTSJ1 mRNA, leading to a premature termination codon at position 597 in exon 9 ( fig. S2A). The c.571+1G>A mutation resulted in an ~80% reduction of FTSJ1 mRNA level in the patient-derived cell line compared to control cell lines ( fig. S2B). Consequently, the 2′-O-methylation modifications at positions 32 and 34 of tRNA Phe were undetectable in the patientderived cell line (Fig. 1D). Similar to Ftsj1 KO mice, loss of 2′-Omethylation led to a decrease in OHyW modification at G37 of tRNA Phe in the patient-derived cells (Fig. 1D). In addition to tRNA Phe , 2′-O-methylation at positions 32 and 34 of tRNA Trp S2C). Upon FTSJ1 expression in patient-derived cells, OHyW modification of tRNA Phe increased slightly (Fig. 1D). The moderate effect of this rescue may be attributed to the poor transfection efficiency of lymphocytes.
We also performed an in vitro methylation assay using recombinant Flag-tagged FTSJ1 and Flag-tagged WD repeat domain 6 (WDR6) WD repeat domain 6 (WDR6), which is the cofactor that binds to FTSJ1 and is required for methylation of position 34 ( fig. S3A) (16). The FTSJ1/WDR6 complex was incubated with hypomodified tRNA Trp purified from patient cells, followed by mass spectrometry analysis. The enzyme complex was sufficient to catalyze 2′-O-methylation of human tRNA Trp in vitro ( fig. S3B).
Loss of Ftsj1 selectively reduces the steady-state level of tRNA Phe in the brain
We examined the transcriptional landscape of tRNAs purified from the forebrain of 8-week-old WT and Ftsj1 KO mice by next-generation sequencing [tRNA sequencing (tRNA-seq)] ( Fig. 2A and fig. S4, A and B). Notably, among all tRNA species, tRNA Phe showed the largest reduction in Ftsj1 KO mouse brain (0.42 fold change relative to WT, P < 0.0001; also see the representative reads in fig. S4B), while other tRNA species only showed small differences between WT and KO mice regardless of the presence or absence of Ftsj1-mediated methylation (0.88 to 1.2 fold change; Fig. 2B). We also examined the profile of the ribosome-bound tRNA from WT and Ftsj1 KO brains ( Fig. 2A) (20). Similar to the total tRNA fraction, ribosome-bound tRNA Phe also showed the largest reduction in the Ftsj1 KO brain when compared to WT brain (0.47 fold change relative to WT, P = 0.0019; Fig. 2C). Notably, in both WT and KO brains, the change in ribosomal tRNA abundance correlated with the change in total tRNA abundance ( fig. S5A).
We also performed Northern blotting to investigate the expression level of tRNA Phe in brain and peripheral tissues, including the liver, kidney, and testis. Similar to tRNA-seq, the steady-state level of tRNA Phe was significantly decreased in the brains of Ftsj1 KO mice (70% of WT brain). The level of tRNA Phe in the liver, kidney, and testis did not show a significant change between WT and KO mice (Fig. 2, D and E). In contrast, the steady-state level of tRNA Leu(CAA) and tRNA Leu(UAA) did not differ between WT and KO mice in any of these tissues (Fig. 2, D and E). We also examined the level of tRNA Phe and tRNA Leu(CAA) in the brain and liver from newborn (P0) mice. The level of tRNA Phe , but not tRNA Leu(CAA) , was decreased in the brain of P0 KO mice ( fig. S5B). Notably, the extent of decrease in tRNA Phe in the Ftsj1 KO brain was moderate (80% of WT brain) at P0 when compared to 8 weeks ( fig. S5C). These results demonstrate that loss of Ftsj1 caused a selective decrease in the abundance of brain tRNA Phe , with the degree of reduction progressing with age.
The selective decrease in tRNA Phe in the Ftsj1 KO brain suggested that hypomodified tRNA Phe is susceptible to fragmentation. Brief incubation of brain lysate without ribonuclease (RNase) inhibitor quickly yielded a short fragment [~35 nucleotides (nt)] of tRNA Phe , which was abundantly accumulated in the Ftsj1 KO mice when compared to WT mice (Fig. 2F). In contrast, the degradation pattern of tRNA Leu(CAA) did not differ between WT and KO brain lysates (Fig. 2F). Next, we extracted small RNA (30 to 40 nt) from WT and Ftsj1 KO brains and performed tRNA-seq. The unbiased analysis showed that both WT and Ftsj1 KO mouse brains contain some extent of tRNA fragments regardless of tRNA species (fig. S5, D and E) (for full dataset, see GSE153758). Notably, the most abundant tRNA fragment accumulated in the KO brain were derived from the 3′ half of tRNA Phe (4.3 fold change relative to WT, P < 0.0001; Fig. 2G and fig. S5D). Furthermore, tRNA-seq of tRNA Phe in the KO mouse brain revealed that a large portion of the 3′ half tRNA Phe fragment was cleaved at position G37, with the rest of the fragment being cleaved at positions 34 to 36 ( fig. S5D).
We also examined whether loss of Ftsj1-mediated 2′-O-methylation affected aminoacylation of tRNA Phe . The level of aminoacylated tRNA Phe in the Ftsj1 KO brain exhibited a marked decrease ( fig. S5F). GmAAOHyWA p m/z 1127.1 (z = -2) GAAOHyWA p m/z 1120.2 (z = -2) with and without Cm, Gm, and OHyW modification. Sequence of each tRNA Phe fragment is shown on the right side of chromatogram. Note that C32, G34, and G37 of tRNA Phe were fully modified to Cm, Gm, and OHyW in WT mouse, whereas Cm and Gm modification was not detected and OHyW showed a ~50% reduction in KO mice. m/z, mass/charge ratio. (D) Modifications of tRNA Phe at C32, G34, and G37 in XLID patient-derived lymphocytes and control lymphocytes from his parents. In the patient-derived lymphocytes, Cm and Gm modifications were absent, and OHyW modification reduced to 12.6% of levels in control cells. Transfection of human FTSJ1 cDNA in the patient-derived cells partially restored Cm, Gm, and OHyW modifications. n.d., not detected. However, the relative aminoacylation level of tRNA Phe in the brain, liver, and kidney, which is calculated by normalizing aminoacylated tRNA Phe to nonaminoacylated tRNA Phe in each tissue, did not differ between WT and KO mice ( fig. S5G). Neither the steady-state level of aminoacylated tRNA Leu(UAA) nor the relative aminoacylation level of tRNA Leu(UAA) differed between WT and KO mice ( fig. S5, H and I). These results suggest that the decrease in aminoacylated tRNA Phe in the Ftsj1 KO brain can be attributed to the decrease in the steadystate level of tRNA Phe , but not to aminoacylation efficiency.
Translational perturbation at the Phe codon in Ftsj1 KO brain We next investigated whether decoding was impaired at Phe codons by ribosome profiling analysis of ribosome-protected mRNA footprints Relative abundance of individual tRNA in the KO mouse brain. tRNA Phe of the KO mouse brain showed the most pronounced decrease compared to that of WT mouse brain (n = 3 for each, ****P < 0.0001 by Student's t test). (C) Relative abundance of individual ribosome-bound tRNA. Among all tRNAs in the KO mouse brain, tRNA Phe showed the largest reduction (n = 3 for each, **P = 0.0019 by Student's t test). (D) Northern blotting of tRNA Phe , tRNA Leu(CAA) , and tRNA Leu(UAA) from total RNA isolated from the brain, liver, kidney, and testis of 8-week-old WT and KO mice. 5.8S ribosomal RNA (rRNA) was used as loading control. (E) Quantitative analysis of tRNA Phe , tRNA Leu(CAA) , and tRNA Leu(UAA) level in indicated tissues relative to the level in the WT brain. tRNA Phe was selectively and significantly decreased in the brain, but not in the liver, kidney, and testis (n = 4 for each, *P = 0.02 by Student's t test). n.s., not significant. (F) Lysates of WT and KO mouse brains were briefly incubated on ice and subjected to RNA extraction and Northern blotting against tRNA Phe and tRNA Leu(CAA) . 5.8S rRNA was used as loading control. Note that a 3′ fragment of tRNA Phe was strongly detected in KO mouse brain lysate. (G) Relative abundance of tRNA fragments in WT and KO mouse brains were analyzed by tRNA-seq. Note that the tRNA Phe -derived fragment was the most abundant tRNA species that accumulated in the KO mouse brain when compared to WT (n = 3 for each, ****P < 0.0001 by Student's t test). Error bars present SEM.
from WT and KO mouse brains at 8 weeks of age ( Fig. 3A) (21). The relative frequency of each codon (herein referred to as ribosome occupancy) of Phe UUC and UUU codons at the ribosomal A site exhibited the highest up-regulation among all codons in the Ftsj1 KO brain (UUC, 1.36-fold, P = 0.0003; UUU, 1.31-fold compared to WT, P = 0.0024) (Fig. 3B). When the relative change of ribosome occupancy was plotted against the relative abundance of ribosomebound tRNA, ribosome occupancy at any given codon, except Phe codons, was positively correlated with the abundance of ribosome bound tRNA (P = 0.0062, Pearson r = 0.3583) (Fig. 3C).
We also performed ribosome profiling using P0 mouse brain and liver ( fig. S6, A and B). In the Ftsj1 KO mouse brain, the Phe UUU codon showed the highest up-regulation of ribosome occupancy among all codons (1.20-fold compared to WT, P = 0.02), whereas the Phe UUC codon only showed 1.05-fold up-regulation (P = 0.6) ( fig. S6A). In contrast, ribosome occupancy at Phe UUU and UUC codons only showed slight or no change, respectively, in the Ftsj1 KO liver when compared to WT (UUU, 1.07-fold, P = 0.009; UUC, 1.01-fold, P = 0.87) ( fig. S6B). When ribosome occupancy at Phe codons in all genotypes and tissues (brain and liver) were plotted to the corresponding tRNA Phe levels, ribosome occupancy at the UUU codon was significantly and negatively correlated with abundance of tRNA Phe (Spearman r = −0.66, P = 0.0121; Pearson r = −0.86, P < 0.0001), whereas the correlation between ribosome occupancy at the UUC codon and tRNA Phe abundance was ambiguous (Spearman r = −0.31, P = 0.29; Pearson r = −0.68, P = 0.0073) (Fig. 3D). Together, these results suggest that loss of Ftjs1 selectively perturbed decoding at Phe codons and that the degree of perturbation may be influenced by the abundance of tRNA Phe and the type of codon.
Defective translation in Ftsj1 KO brain and its link to brain dysfunction Dysregulation of decoding can affect gene expression at both transcriptional and translational levels. We performed RNA-seq using WT and Ftsj1 KO mouse brains at P0 and 8 weeks of age and compared the transcriptome (Fig. 3A). At both developmental stages, only a small subset of genes showed change at the mRNA level in Ftsj1 KO mouse brain, and the degree of change was small compared to WT (fig. S6, C to F). Among genes that showed significant changes (P < 0.01) in the Ftsj1 KO brain at P0 stage, 27 genes showed a 0.72-to 0.88-fold decrease, and seven genes showed a 1.11-to 1.25-fold increase compared to the WT brain at P0 ( fig. S6C). Gene ontology analysis of differentially regulated genes revealed that these genes are involved in metabolic processes not specifically limited to neuronal function ( fig. S6D). Likewise, among genes that showed significant changes (P < 0.01) in 8-week-old KO mouse brain, 55 genes showed 0.7-to 0.9-fold decrease, and 19 genes showed 1.09-to 1.40-fold increase compared to the WT brain ( fig. S6E). The down-regulated genes were enriched in pathways that affect neuronal development and structure, while the up-regulated genes had only limited enrichment in pathways related to neuron functions ( fig. S6F).
We next estimated TE based on RNA-seq and ribosomal profiling data, a measure that is generally in proportion with protein translation rate (Fig. 3A). TE was determined by dividing the normalized read number of ribosome-bound mRNA footprints to normalized read number of the corresponding mRNA for each gene. We selected 4474 genes from datasets from P0 brain and 5577 genes from datasets from 8-week-old brain based on the following criteria: (i) Genes are protein-encoding genes, and (ii) genes are consistently detected across WT and KO brain samples (three biological replicates each) at each developmental stage. The mean TE of gene products from the KO brain was significantly lower than that of the WT brain at 8 weeks (WT, 1.452; KO, 1.428; P = 0.0048), while the mean TE did not differ between WT and KO brains at P0 (WT, 2.077; KO, 2.070, P = 0.63) (Fig. 3E). Deficiency of Trm7 in yeast induced a strong general amino acid control (GAAC) pathway and is involved in the growth defect (22). However, genes related to the GAAC pathway in mammals did not show a significant increase in the Ftsj1 KO mouse brain at both 8 weeks of age and P0 stage ( fig. S6G), as well as mutant lymphocyte cells derived from the patient ( fig. S6H).
We performed gene ontology analysis on genes that showed significant (P < 0.01) changes in TE at both P0 and 8 weeks of age. In P0 KO mouse brain, 74 genes showed a 0.38-to 0.84-fold decrease in TE, and 70 genes showed a 1.20-to 4.09-fold increase in TE compared to the WT brain ( fig. S6I). These differentially changed genes in P0 KO brain were not specifically related to neuronal functions ( fig. S6J). On the other hand, in 8-weeks-old KO mouse brain, 109 genes showed a 0.28-to 0.75-fold decrease in TE, and 80 genes showed a 1.34-to 3.26-fold increase in TE (Fig. 3F). The translationally down-regulated genes are actively involved the brain functions including cell projection, neuronal development, and synapse organization and correspond to membrane and synaptic proteins (Fig. 3, G and H). In contrast, the translationally up-regulated genes are not enriched in any particular biological processes ( fig. S6K).
A close inspection of the translationally down-regulated genes revealed that some are involved in Wnt signaling [e.g., Ctnnb1 (-catenin 1), Wnt7b, and Pak2] (Fig. 3I) and are important for brain development and maturation (23)(24)(25). Notably, deficiency of Ctnnb1 has been implicated in neurological disorders including ID and autism in humans (26,27). Furthermore, genes related to neuron adhesion or synaptic spine formation, such as tenascin R (Tnr) and discs large MAGUK scaffold protein 2 (Dlg2), also exhibited a decrease in TE in 8-week-old KO mouse brain. Deficiencies of these genes have been implicated in cognitive deficits in animal models (28,29). The 109 genes with significantly down-regulated TE in the KO mouse brain belong to a group of genes that are efficiently translated in the WT mouse brain (mean TE of the 109 genes in WT: 2.326 versus mean background TE of WT: 1.442, P < 0.0001; fig. S6L).
Altered neuron ultrastructure in the hippocampus and cortex of Ftsj1 KO brain
We next examined the morphology of cortex and hippocampus at histological, cellular, and synaptic levels using 8-week-old mouse brain. Nissl staining revealed that the macroscopic morphology of cortex and hippocampus was comparable between WT and KO mice ( fig. S7, A and B). There was no obvious difference in the population of hippocampal and cortical neurons and glial cells between WT and KO mice ( fig. S7, C to E). We also performed histological examination of the liver, kidney, heart, and testis by hematoxylin and eosin (H&E) staining; however, there were no morphological changes in these tissues in KO mice ( fig. S7F).
We also investigated the morphology of dendritic spines in cortical and hippocampal neurons by Golgi staining. Dendritic spine ultrastructure and density have been implicated in many aspects of brain functions including learning and memory (30). Dendritic spines can be categorized into distinct types depending on their shape, which has been correlated with neuronal function. In 8-week-old Ftsj1 KO brain, both hippocampal and cortical neurons exhibited a significant increase in thin dendritic spines and a concomitant decrease in mushroom type spines when compared to the WT brain, an indication of immature spine formation (Fig. 4, A and B). In addition, we also examined striatal neurons that are also implicated learning and memory (31,32). Ftsj1 KO mice exhibited an increase in the immature filopodia-like spines and a concomitant decrease in mushroom-type spines when compared to the WT brain ( fig. S7, G and H). Moreover, the density of dendritic spines was significantly decreased in Ftsj1 KO hippocampal neurons compared to WT (Fig. 4C). We also analyzed the morphology of the postsynaptic density (PSD) using electron microscopy (Fig. 4D). In both hippocampal and cortical neurons, there was a significant decrease in the length and width of the PSD (Fig. 4, E and F).
Impaired long-term potentiation and long-term depression in the hippocampus of Ftsj1 KO mice
We performed electrophysiological analysis using hippocampal slices prepared from WT and Ftsj1 KO brains from mice at 8 weeks of age. A paired-pulse facilitation protocol was applied to the hippocampal slices to examine the presynaptic release of neurotransmitter by measuring the relative increase in population excitatory postsynaptic potential (pEPSP) (fig. S7I). The amplitude of facilitation did not differ between WT and Ftsj1 KO mice ( fig. S7J). We also investigated the relationship between the intensity of presynaptic stimulus input with the pEPSP output (I/O curve) using the same hippocampal slices ( fig. S7K). There was no significant difference of I/O curve between WT and KO mice (fig. S7L). These results suggest that neurotransmitter release and its corresponding postsynaptic response were well preserved in the Ftsj1 KO brain at the basal level. Repeated electrical stimuli can produce a persistent increase or decrease in the efficiency of synaptic transmission known as longterm potentiation (LTP) or long-term depression (LTD), respectively. LTP and LTD are associated with learning and memory and are mediated by complex molecular signaling including transcriptional, posttranscriptional, translational, and posttranslational mechanisms. We found that theta burst stimulation (TBS) induced a robust LTP in WT mouse hippocampal slices (Fig. 5, A and B). In hippocampal slices from KO mice, the normalized pEPSP slope of TBS-evoked LTP showed a 50% reduction compared to WT (WT, 1.66 ± 0.05; KO, 1.33 ± 0.06; ***P < 0.0001, WT versus KO; Fig. 5C).
In LTD experiments, the low-frequency stimulation (LFS) effectively suppressed the pEPSP slope in hippocampal slices of the WT mouse brain (0.75 ± 0.02 related to baseline; Fig. 5, D to F). The LFS induced only a slight decrease in the pEPSP slope in hippocampal slices of the KO mouse brain (0.90 ± 0.02 related to baseline; WT versus KO, ***P < 0.001; Fig. 5, D to F), suggesting that LTD formation was impaired in the hippocampus of the KO mouse. Notably, both LTP and LTD were effectively blocked by application of D-2-amino-5phosphonopentanoate (AP5), an inhibitor of the N-methyl-d-aspartate (NMDA) receptor, in WT and KO hippocampal slices (Fig. 5, C and F).
Ftsj1 KO mice show behavioral abnormality
We performed a battery of behavioral tests in 8-week-old WT and KO mice. First, mice were subjected to open-field and elevated plus maze tests to examine locomotor activity and anxiety-like behavior in a novel environment (Fig. 6A) because anxiety is a symptom observed in some patients with FTSJ1-related XLID (9). Ftsj1 KO mice spent less time and traveled less distance in the center of the openfield chamber than WT mice, while the total travel distance in the chamber did not differ between WT and Ftsj1 KO mice (Fig. 6A). In the elevated plus maze, Ftsj1 KO mice spent less time and traveled less distance in the open arm than WT mice, while the total travel distance in both open arm and closed arm was longer in KO mice than in WT (Fig. 6B). The results from the open-field and elevated plus maze tests suggest that loss of Ftsj1 induced anxiety-like behavior in mouse.
Next, we investigated whether loss of Ftsj1 would affect spatial learning using the Barnes maze test, in which mice were trained to find a hidden chamber using spatial cues as guidance (Fig. 6C). The number of mistakes until the mouse found the goal chamber was counted as a primary error number to evaluate the ability of spatial leaning. Compared to WT mice, Ftsj1 KO mice showed slower spatial learning, with the primary error number being significantly higher than WT mice in the first and second day of training (Fig. 6D).
Last, mice were subjected to a fear conditioning paradigm to assess how loss of Ftsj1 would affect the formation and retrieval of fear memory. During the conditioning day, WT and Ftsj1 KO mice showed the same degree of freezing responses to the mild foot shock and sound presentation (Fig. 6E). On the next day, the retrieval of fear memory was tested by putting mice in the chamber where they received foot shock (context) and was placed in a new chamber with sound presentation (cue) that they had heard on the conditioning day. Compared to WT mice, Ftsj1 KO mice showed significantly less freezing behavior in response to the context-dependent stimuli and the cue-dependent stimuli (Fig. 6, F and G).
DISCUSSION
Defective tRNA modifications feature in a number of brain disorders (3,(33)(34)(35). To date, at least 17 genes encoding cytosolic tRNA modification enzymes, including FTSJ1, have been identified as causal genes for intellectual disabilities, microcephaly, and neurodegeneration (3). However, given their ubiquitous presence throughout the body, understanding why loss of these modifications preferentially affects brain function remains a challenge. In the present study, we have investigated the impact of Ftsj1-deficiency in the mouse brain and uncovered a link to the pathogenesis of FTSJ1-related XLID. Among all tRNA species, hypomodified tRNA Phe was selectively and significantly reduced in the Ftsj1 KO mouse brain, but not in other tissues. Furthermore, decoding at Phe codons was selectively and markedly impaired in the Ftsj1 KO brain, leading to a decrease in TE of genes related to synaptic activity and structure. Thus, our results suggest that the selective decrease in hypomodified tRNA Phe in the brain is associated with the nonsyndromic FTSJ1-related XLID.
Hypomodified tRNA Phe can be susceptible to endoribonucleasemediated cleavage, which may lead to a decrease in its steady-state level. When preparing RNA without RNase inhibitor, we observed an accumulation of 3′-tRNA Phe fragment in the Ftsj1 KO mouse brain. Concordant with our result, loss of Drosophila Ftsj1 homologs also resulted in an accumulation of 3′-tRNA Phe fragment, although the steady state of full-length tRNA Phe did not change in Drosophila (18). RNase 1 and angiogenin/RNase 5 are the members of ribonuclease A (RNase A) family responsible for the cleavage of tRNA, and their actions can be blocked by 2′-O-methylation of ribose and methylation at nucleotide base. For example, tRNA Met contains 2′-O-methylation at position C34, which is mediated by nucleolar fibrillarin and small nucleolar RNAs (36). Hypomodified tRNA Met at C34 is highly sensitive to angiogenin-mediated cleavage, which efficiently induced generation of 3′-tRNA Met fragment (36). In addition, NOP2/Sun RNA methyltransferase 2 (NSUN2) is a cytosine C5-methyltransferase for both tRNA and mRNA, and a loss-of-function mutation in NSUN2 has been identified in patients with ID (37)(38)(39). Notably, loss of NSUN2 resulted in RNase 5/angiogenin-mediated cleavage of hypomodified tRNA, which is possibly involved in the pathogenesis (40). Thus, an RNase A ribonuclease family member is likely involved in the cleavage of hypomodified tRNA Phe in Ftsj1 KO mice. Identification of the responsible RNase in future studies will be important for the development of a therapeutic target.
The cause of brain-specific decrease in tRNA Phe is unclear but might result from tissue-specific differences in the balance of RNase and RNase inhibitor. In mouse and human brain, the major RNase A family member is RNase 1, which has moderate levels of mRNA expression compared to other tissues (41). On the other hand, RNase/angiogenin inhibitor 1, the endogenous inhibitor of both RNase 1 and angiogenin (42,43), is expressed at lower levels in the brain compared to other tissues such as the liver and pancreas (41). The high RNase-to-RNase inhibitor ratio suggests that the brain contains a relatively high RNase activity, which subsequently induces degradation of hypomodified tRNA Phe . This degradation may lead to the manifestation of ID without other physical abnormalities. In addition, the potentially high brain RNase activity might explain why deficiencies of other cytosolic tRNA modification enzymes such as NSUN2 and elongator acetyltransferase complex subunit 2 (ELP2) also lead to the development of brain malfunction as the primary symptom (40,44). Among the 11 Ftsj1 substrate tRNAs, we found that tRNA Phe showed the highest sensitivity to endoribonuclease-mediated cleavage in the Ftsj1 KO mouse brain, despite the fact that 2′-O-methylation was eliminated from positions 32 and 34 of these tRNAs. The reason for the selective cleavage of tRNA Phe , but not other Ftsj1 substrate tRNAs, is currently unknown. One possible explanation may be that the hypermodified G37 of tRNA Phe is associated with loss of the OHyW modification, which is exclusively found at G37 of tRNA Phe and is the bulkiest modification found at position 37 of all Ftsj1 substrate tRNAs. It is conceivable that OHyW modification may prevent access of RNase to tRNA Phe through spatial interference. Consistent with this hypothesis, our tRNA-seq showed that a majority of tRNA Phe fragments found in Ftsj1 KO mouse brain were cleaved at the hypomodified G37. Further studies using OHyWdeficient cells are needed to determine the extent to which loss of OHyW modification induces tRNA Phe cleavage. tRNA Phe , along with tRNA Trp and tRNA Leu(UAA) , is the only three tRNAs that are 2′-O-methylated at both positions 32 and 34 by Ftsj1. Accordingly, tRNA Phe is the only Ftsj1 substrate tRNA that is hypomodified at all three positions (32, 34, and 37) upon Ftsj1 deficiency. Thus, a collective loss of both 2′-O-methylation and OHyW modification may cause the selective cleavage of tRNA Phe in Ftsj1 KO mouse.
Our data from ribosome profiling of the brain at different developmental stages provide clear evidence that ribosome transition is selectively impaired at Phe codons in the Ftsj1 KO mouse brain. Defective decoding at Phe codons was selectively observed in the Ftsj1 KO mouse brain, in which tRNA Phe levels were significantly decreased. These results suggest that the decoding efficiency at Phe codons depends on the quantity of tRNA Phe . Note that decoding at the Phe UUU codon was impaired in Ftsj1 KO mouse brain at both adult and neonatal stages, while decoding at the Phe UUC codon was only impaired at the adult stage. Because tRNA Phe forms wobble binding (G:U) with the third position of the UUU codon and the affinity of tRNA Phe :UUU base pairing is relatively low compared to tRNA Phe :UUC base pairing, it is conceivable that decoding at the Phe UUU codon is more susceptible to the steady-state tRNA Phe level than decoding at the Phe UUC codon. Note that defective modifications of tRNA Phe might directly impair codon-anticodon binding, further contributing to the decoding defect. Previous studies have shown that yW modification at G37 of tRNA Phe is also involved in codon-anticodon recognition (45). Thus, the slow ribosomal transition at Phe codons might be attributed to both quantitative and qualitative defects of hypomodified tRNA Phe in the Ftsj1 KO mouse brain.
Here, we also present a thorough transcriptome and the translational profile from the brain. A previous study has shown that Trm7-deficient yeast exhibited a marked increase in the transcription of genes related to amino acid metabolism induced by Gcn4, the transcription factor related to nutrient stress response (46). In contrast, the Ftsj1 KO mouse brain showed only a limited change in the overall transcriptome, including amino acid metabolism while exhibiting a marked rearrangement of TE. Notably, the TE of a subset of genes related to maintenance of synaptic activity and structure was markedly reduced in the Ftsj1 KO mouse brain. Consequently, Ftsj1 KO mouse hippocampus and cortex exhibited immature spine morphology and decreased synaptic plasticity, ultimately leading to impairment of learning and memory and anxiety-like behavior. These results thus provided molecular, histological, and physiological links to ID related to Ftsj1 deficiency. Other brain regions, such as the amygdala and cerebellum, are also involved in the learning and memory (47,48) and may also be affected by Ftsj1 deficiency. A detailed and broad inspection of brain region-specific neuron morphology and function will be conducted in future studies.
Note that in addition to the brain dysfunction, Ftsj1 KO mice exhibited a moderate reduction in body weight, which was not apparent in patients having FTSJ1 deficiency (8,12). Similar to our findings in mice, reduced body weight was observed in Drosophila with deficiency of Ftsj1 homologs (18) and a line of Ftsj1 gene-trapped mice (19). These findings suggest that Ftsj1 might have a broad impact on peripheral tissues. The molecular mechanism underlying the low body weight in mouse and Drosophila is currently unknown. Similar to mouse peripheral tissues, the steady-state level of hypomodified tRNA Phe remains unchanged in Drosophila (18). It is conceivable that the hypomodification of tRNA Phe and other Ftsj1 substrate tRNAs may impair general translational efficiency by affecting base pairing independent of the steady-state tRNA Phe level.
In this case, the reduction of general protein translation levels could result in decreased cell growth and cell mass, ultimately leading to reduced body weight. Given the ubiquitous expression of Ftsj1, future studies will be needed to elucidate the general role of Ftsj1 in non-neuronal tissues in eukaryotes.
Animals
A constitutive Ftsj1 KO mouse line was generated by conventional homologous recombination to replace exons 2 to 5 of the Ftsj1 gene with the neomycin resistance gene (see fig. S1A). Ftsj1 heterozygous female mice were mated to WT littermate male mice to obtain male Ftsj1 KO mice and their littermate controls. Mice were housed at 25°C with 12-hour light and 12-hour dark cycles. To obtain P0 mice, a pair of female and male mice were housed in the same cage for 1 hour (19:00 to 20:00). Subsequently, female mice were placed in a new cage and monitored carefully during pregnancy. When female mice gave birth in the night (20:00 to 7:00), P0 mice were taken the next morning for genotyping and experiments as P0 samples. The Animal Ethics Committee of Kumamoto University reviewed and approved all animal procedures (approval ID: A2019−107 R2).
Human cells
Immortal lymphoblast cells were established from blood samples of a Japanese family as described (12). These blood samples were obtained with informed consent approved by Institute Review Board, National Center of Neurology and Psychiatry (XXXX-115). Control cell lines #1 and #2 were derived from peripheral blood cells of the patient's father and mother, respectively. The father carried the normal FTSJ1 gene, while the mother was a heterozygous carrier of the c.571+1G>A mutation of the FTSJ1 gene [see (12) for detailed information]. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, and RNA was extracted for quantitative polymerase chain reaction (PCR) and mass spectrometry analysis. Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and were used for exogenous expression of FTSJ1-Flag, WDR6-V5, and THADA armadillo repeat containing (THADA)-V5.
Genotyping
To genotype the Ftsj1 KO mice, genomic DNA was extracted from small pieces of tails clipped from 4-week-old mice or P0 mice using phenol/chloroform precipitation. Ten nanograms of DNA was subjected to PCR for genotyping using primers to detect the neomycin resistance gene.
Protein-protein interaction analysis
The expression vectors of FTSJ1-FLAG and WDR6-V5 (1 g each per well in a six-well plate) were cotransfected into HEK293T cells (2.5 × 10 5 cells per well) using polyethyleneimine (51). After 48 hours, the cells were collected and lysed with 0.5 ml of lysis buffer [150 mM KCl, 10 mM tris-HCl (pH 8.0), 2.5 mM MgCl 2 , 1 mM dithiothreitol (DTT), 0.5% Triton X-100, and 1× complete EDTAfree protease inhibitor cocktail (Roche)] by 20 passages through a 25-gauge syringe needle. The lysate was centrifuged twice at 20,000g for 20 min at 4°C and passed through an Ultrafree 0.45-m filter (Merck Millipore) to remove cell debris. Immunoprecipitation was conducted by incubating the supernatant with 20 l of anti-Flag M2 affinity gel (Sigma-Aldrich) for 3 hours at 4°C and washing the gel by lysis buffer three times. The gel was boiled at 95°C for 5 min for SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted using anti-Flag-tag polyclonal antibody conjugated with horseradish peroxidase (HRP) (Anti-DDDDK-tag pAb-HRP-DirecT, Medical & Biological Laboratories), or anti-V5-tag polyclonal antibody conjugated with HRP (Anti-V5-tag pAb-HRP-DirecT, Medical & Biological Laboratories) diluted with Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo) at 1:4000 (v/v). Chemiluminescence was detected using Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific) and an ImageQuant LAS4000 mini system (GE Healthcare).
Histology and electron microscopy
Ftsj1 KO and WT mice were euthanized and perfused with 4% paraformaldehyde for histological examination. H&E staining and Nissl staining was performed according to standard protocols. Golgi staining of hippocampal and cortical neurons was performed using the FD Rapid GolgiStain Kit (FD NeuroTechnologies Inc.). Images were acquired using an IX83 Inverted Microscope (Olympus). For immunofluorescent staining, brain slices were stained with anti-Map2 and glial fibrillary acidic protein antibodies, followed by incubation with secondary antibodies conjugated with Alexa Fluor 488. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Images were acquired using a confocal microscope (Olympus FV3000). For transmission electron microscopy, mice were fixed with 2% paraformaldehyde and 2% glutaraldehyde, and hippocampal regions were dissected for downstream processing. Random sections were obtained, and images at a ×5000 magnification were used to calculate the length and the width of the PSD with ImageJ software.
Northern blot
The brain, liver, kidney, and testis of Ftsj1 KO mice or WT littermates were collected, snap-frozen in liquid nitrogen, and stored at −80°C. Right half-brains were used for tRNA and aminoacyl-tRNA analysis, and left half-brains were used for ribosome profiling. Each frozen right half-brain was transferred into 5 ml of TRI Reagent (Molecular Research Center) and homogenized using TissueRuptor (QIAGEN), followed by centrifugation at 12,000g for 10 min at 4°C to remove debris. The brain lysate in TRI Reagent was split into two, one for conventional tRNA Northern blot and the other for aminoacyl-tRNA analysis by acidic Northern blot. For conventional tRNA Northern blot, RNA was extracted according to the manufacturer's protocol. The liver, kidney, and testis were also homogenized in TRI Reagent, and RNA extraction was performed in the same way as brain. For brain lysate tRNA cleavage analysis, brain lysate was prepared as described in the ribosome profiling method section, and the lysate was incubated on ice for 10 min, followed by RNA extraction using TRI Reagent. For conventional Northern blot, total RNA was resolved using denaturing 7 M urea/tris-borate EDTA/PAGE, and Northern blot was performed using DNA oligo probes that were Digoxigenin (DIG)-labeled using DIG oligonucleotide 3′-end labeling kit second generation (Roche). Aminoacyl-tRNA analysis was performed essentially as described (52). Briefly, after the total RNA pellet was collected from tissue lysate in TRI Reagent by isopropanol precipitation, the RNA pellet was rinsed with 75% ethanol/10 mM Sodium acetate (pH 5.0)/1 mM EDTA and dissolved in 10 mM NaOAc (pH 5.0)/1 mM EDTA. Aminoacylation level was monitored using acidic PAGE/Northern blot, using the same DIG-labeled probes as conventional Northern blot. The sequences of oligo DNA probes are as follows: mouse/human tRNA Phe , 5′-CGAAACCCGGGATCGAACCAGGGACCTTTA; mouse/human tRNA Leu(CAA) , 5′-TGTCAGAAGTGGGATTC-GAACCCACGCCTC; mouse/human tRNA Leu(UAA) , 5′-TAC-CAGAAGTGGGGTTCGAACCCACGCGGA; mouse/human 5.8S rRNA, 5′-GCAAGTGCGTTCGAAGTGTCGATGATCAAT.
Ribosome profiling
To analyze ribosome-contained mRNA fragments, steps such as RNase I treatment, ribosome recovery, footprint fragment purification, 3′ adaptor ligation, rRNA depletion, reverse transcription, circularization, and PCR amplification were performed essentially as described (21), with some modifications as described below. Brain or liver lysis was performed by transferring each frozen left halfbrain or liver into polysome buffer (20) and homogenizing using TissueRuptor (QIAGEN). For 3′ adaptor ligation, air-adenylated linker A (BIOO, 5rApp/CTGTAGGCACCATCAAT/3ddC/) was used. cDNA libraries of ribosome-contained mRNA fragments were initially sequenced using MiSeq to perform A site codon analysis using Galaxy (53) and RiboGalaxy (54): First, fastq files were filtered by quality, the 3′ adaptor sequence (CTGTAGGCACCAT-CAAT) was removed, and rRNA reads were removed by Bowtie2. Ribosome-contained mRNA fragment sequences were acquired by aligning reads to mouse RefSeq mRNA sequences and were analyzed using triplet periodicity and metagene analysis programs in RiboGalaxy, to determine the appropriate footprint length and codon frames. For large-scale ribosome-contained mRNA fragment analysis, cDNA libraries were subjected to Illumina HiSeq2500 sequencing. Using Galaxy, fastq files were filtered by quality, the adaptor sequence (CTGTAGGCACCATCAAT) was removed, and mRNA reads were retrieved by aligning to mouse RefSeq mRNAs. mRNA reads were aligned, counted, and analyzed using HISAT2, HTSeq-count, and DESeq2. Gene ontology analysis was performed using Gene Set Enrichment Analysis (GSEA) website software (55).
Transcriptome analysis
To collect brain or liver total RNA, immediately following ribosome profiling tissue lysis, a fraction of tissue lysate was mixed with TRI Reagent, and total RNA was extracted. Polyadenylate [poly(A)] + RNA was collected from total RNA using the oligotex-dT30 Super mRNA Purification Kit (TaKaRa). Poly(A) + RNA was fragmented and collected by incubation in alkaline buffer (88 mM NaHCO 3 , 12 mM Na 2 CO 3 , and 1 mM EDTA) at 95°C for 45 min, followed by ethanol precipitation. Poly(A) + RNA fragments were resolved by denaturing urea PAGE, and fragments of 20 to 40 nt were gelexcised and extracted. 3′ adaptor ligation, library preparation, and sequencing were performed in the same way as ribosome profiling. Using Galaxy (53), fastq files were filtered by quality, 3′ adaptor sequence (CTGTAGGCACCATCAAT) was removed, and mRNA reads were retrieved by aligning to mouse RefSeq mRNAs. mRNA reads were aligned, counted, and analyzed using HISAT2, HTSeqcount, and DESeq2. tRNA and tRNA fragment (tRF) sequencing Total tRNA, ribosome-contained tRNA, and tRF sequencing analyses were performed essentially as described (20). For total tRNA and tRF preparation, total RNA was first prepared from brain lysate (also used for ribosome profiling and transcriptome analysis) using TRI Reagent. Total RNA was resolved by denaturing urea PAGE, and tRNA (60 to 100 nt) and tRF (20 to 40 nt) were collected by gel excision. To collect ribosome-contained tRNA, during gel excision of mRNA fragments (20 to 40 nt) in ribosome profiling, tRNA fragments (60 to 100 nt) were also collected. Total tRNA, ribosome-contained tRNA, and tRF were demethylated using Escherichia coli-derived Alpha-ketoglutarate-dependent dioxygenase (AlkB) to enable reverse transcription (56). Briefly, tRNA or tRF were incubated in 45 mM tris-HCl (pH 8), 0.9 mM -ketoglutaric acid, 1.8 mM ascorbic acid, 67 M (NH 4 ) 2 Fe(SO 4 ) 2 , and 2.5 M AlkB, at 37°C for 2 hours. Demethylation was confirmed by performing yeast tRNA demethylation and RNA nucleoside mass spectrometry analysis, as a positive control. Subsequently, library preparation was performed in the same way as mRNA library preparation. After Illumina MiSeq sequencing, tRNA and tRF analysis was performed using Galaxy. Briefly, fastq files were converted to fasta files, 12 nt of adaptor sequence (GG-CACCATCAAT) was clipped (leaving 5 nt of 3′ adaptor sequence CTGTA at the tRNA 3′ end to allow mapping of short reads), and tRNA reads were Bowtie2-aligned to mouse tRNA sequences in the Genomic tRNA database (57). Reads were counted using alignment position information in the SAM file and count function in Galaxy.
Electrophysiology
WT and Ftsj1 KO mice at 8 weeks old were used for electrophysiological experiments. Briefly, mice were quickly euthanized by cervical dislocation, and mouse hippocampal slices were acutely prepared at 400 m in thickness using a vibratome (Leica). Slices were incubated in artificial cerebrospinal fluid (CSF; 123 mM NaCl, 3 mM KCl, 1.25 mM NaH 2 PO 4 , 2 mM MgSO 4 , 2 mM CaCl 2 , 10 mM glucose, and 26 mM NaHCO 3 ) for 60 min with consistent aeration. A glass micropipette that was filled with artificial CSF was placed on the CA1 region to record the pEPSPs, and a stimulating electrode was placed on the Schaffer collateral fibers. The paired-pulse ratio of the pEPSP response was calculated as the ratio of the second pEPSP to the first pEPSP stimulated at a 50-ms interpulse interval. LTP was induced by TBS consist of five bursts of five pulses with 10-ms intervals, delivered at 200-ms intervals. LTD was induced by LFS that consists of 900-pulse stimulation at 1-Hz frequency.
Behavioral tests
WT and Ftsj1 KO mice were used for a battery of behavioral tests by an experimenter who was blinded to the genotype. Mice at 7 weeks old were handled for 1 week. Mice were then subjected to open-field test, elevated plus maze, Barnes maze, and fear conditioning test with 1-week interval between each test. For open-field test, a 60 cmby-60 cm-by-60 cm chamber with gray color was used. Mice were placed in the center of the chamber, and the exploring behavior was recorded for 10 min using a video camera above the chamber. Video software (Actimetrics) was used to define a six-by-six grid, and the center two-by-two grid was defined as the center area. Total distance moved in the entire area of chamber, distance moved in the center area of chamber, and time spent in the center area of chamber were analyzed.
Elevated plus maze test was performed as described previously (58). The elevated plus maze used in this study consisted of four arms (two open arms and two arms closed by 10-cm-high walls), 30 The Barnes maze analysis was performed as described with modifications (59). The Barnes maze consisted of a circular platform (91 cm in diameter) with 20 equally spaced holes (5 cm in diameter) and was elevated 100 cm above the floor. A black box was located under a hole of the platform so the mouse could escape from the open platform surface. Visual cues (triangle, rectangle, circle, and cross) were placed surrounding the maze. On the trial day, mice were place in a dark start chamber in the middle of the platform. After 10 s, the start chamber was lifted, and the movement of each mouse was recorded for 3 min. The trial ended when the mouse entered the goal in 3 min. When the mouse could not find the black box in 3 min, the trial was then forced to end by gently guiding the mouse to the black box. Mice received four trials per day with an intertrial interval of 20 min. Number of pokes to any hole before entering the black box was considered as an error, and the number of errors was used to assess spatial learning ability (Actimetrics).
Fear conditioning test was performed as described previously with modifications (60). For the conditioning of contextual and cued fear memory, mice were placed in the test box and received three identical trials. A trial consists of 120-s habituation period, followed by an auditory cue at 80 dB for 30 s in combination with a mild foot shock (0.5 mA) during the last 2 s of auditory presentation. Next day, mice were placed in the same test box and were monitored for 6 min without auditory presentation. Mice were then removed from the test chamber and placed in their home cage for 60 min before cue testing. Cue testing was performed in a new test box with the floor being changed to plastic board and the ceiling of the test box being changed to triangle. Mice were allowed to freely explore the box for 3 min, followed by auditory presentation for 3 min at 80 dB without foot shock. Freezing behavior was examined using software (Actimetrics) following the instruction.
Quantification and statistical analysis
Data were analyzed using GraphPad Prism 8 software. Unpaired Student's t tests or Mann-Whitney tests were used to assess differences between two groups, and two-way analysis of variance (ANOVA) tests followed by Tukey's multiple comparisons were used to examine differences among multiple groups. A two-tailed P value of 0.05 was considered significant unless specified. Data are presented as means ± SEM.
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/7/13/eabf3072/DC1 View/request a protocol for this paper from Bio-protocol. | 2021-03-27T13:08:26.882Z | 2021-03-01T00:00:00.000 | {
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263937306 | pes2o/s2orc | v3-fos-license | Atomistic investigation of an Iowa Amyloid-β trimer in aqueous solution
The self-assembly of Amyloid beta (Aβ) peptides are widely accepted to associate with Alzheimer's disease (AD) via several proposed mechanisms. Because Aβ oligomers exist in a complicated environment consisting of various forms of Aβ, including oligomers, protofibrils, and fibrils, their structure has not been well understood. The negatively charged residue D23 is one of the critical residues of the Aβ peptide as it is located in the central hydrophobic domain of the Aβ N-terminal and forms a salt-bridge D23-K28, which helps stabilize the loop domain. In the familial Iowa (D23N) mutant, the total net charge of Aβ oligomers decreases, resulting in the decrease of electrostatic repulsion between D23N Aβ monomers and thus the increase in their self-aggregation rate. In this work, the impact of the D23N mutation on 3Aβ11–40 trimer was characterized utilizing temperature replica exchange molecular dynamics (REMD) simulations. Our simulation reveals that D23N mutation significantly enhances the affinity between the constituting chains in the trimer, increases the β-content (especially in the sequence 21–23), and shifts the β-strand hydrophobic core from crossing arrangement to parallel arrangement, which is consistent with the increase in self-aggregation rate. Molecular docking indicates that the Aβ fibril-binding ligands bind to the D23N and WT forms at different poses. These compounds prefer to bind to the N-terminal β-strand of the D23N mutant trimer, while they mostly bind to the N-terminal loop region of the WT. It is important to take into account the difference in the binding of ligands to mutant and wild type Aβ peptides in designing efficient inhibitors for various types of AD.
Introduction
World Health Organization (WHO) reported that dementia affected approximately 50 million people worldwide in 2017 and the number of new patients could increase to ca. 10 million annually. 1 Alzheimer's disease (AD) is the most common type of dementia (60-70% cases), 1 which is strongly linked to the self-aggregation of the Amyloid beta (Ab) peptides. 2 Ab peptides consist of 36-43 residues formed via the proteolysis of the transmembrane Amyloid Precursor Protein (APP). According to the Amyloid cascade hypothesis, 3-5 Ab peptides self-assemble into Ab oligomers, which have been shown to be neurotoxic via several mechanisms, including damaging neurites and synapses 2,6 or forming transmembrane pores that disrupt Ca 2+ homeostasis. 7,8 The most abundant form of Ab peptides consists of 40 amino acids (Ab 40 ) and accounts for approximately 80-90% of the total population. 9 The development of AD therapy relies on the understanding of Ab oligomer structures, [10][11][12][13] unfortunately, because Ab oligomers exist in complicated environments comprising of numerous forms of oligomers and brils, their structure has not been well characterized. 11,14 Therefore, molecular dynamics simulation has been one of the main tools in assessing the structure of Ab oligomers. [15][16][17] The self-assembly process of Ab peptides highly depends on their amino acid sequence. 18 Mutations can alter the Ab oligomer structures and their properties. Especially, the common mutations in the central hydrophobic domain of the Ab Nterminal, including Flemish (A21G), 19 Dutch (E22Q), 20 Italian (E22K), 21 Arctic (E22G), 22 and Iowa (D23N) 23 signicantly affect the dynamics and conformations of the Ab oligomers and brils. It is known that the central hydrophobic domains of an Ab chain forms rigid b-structure that in turn forms strong interaction with other Ab chains, leading to Ab selfaggregation. Inhibitors that target the N-terminal b-strand have been shown to successfully inhibit Ab selfaggregation. [24][25][26] In the N-terminal hydrophobic domain, D23 stabilizes the loop region of Ab peptides by forming a salt-bridge to the residue K28 and polar contacts to other residues in loop domain. [27][28][29] Substituting the negatively charged residue D23 by another residue could signicantly alter the structure of Ab peptides. In the familial Iowa mutant, D23 is replaced by the neutral residue asparagine, 23 which reduces the total net charge and thus the electrostatic repulsion between constituting chains of Ab oligomers. Consequently, the self-aggregation rate of D23N oligomers has been shown to increase. 30 Therefore, studying the impact of the D23N mutation on the self-assembly has been carried out for a number of Ab peptide systems, including Ab 21-30 , 31 Ab 10-35 , 32 Ab 1-40 , 33 Ab 1-42 , 34 2Ab 1-40 , 35 and 6Ab [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] . 36 However, the effect of D23N mutation on Ab 40 trimer (3Ab 40 ), one of the most neurotoxic Ab oligomers, 10,11 has not been revealed.
We are interested in Ab 40 as it is the most abundance form of Ab. Although Ab 42 oligomers are more neurotoxic than Ab 40 oligomers, the population of Ab 40 peptides is approximately ten times higher than that of Ab 42 . 9 Thus, structural investigation of Ab 40 and Ab 42 are both important. In addition, structure-neurotoxicity relationship has been established for Ab 40 oligomers but not for Ab 42 oligomers. 37 In order to gain insights into the changes in the structure and dynamics of the neurotoxic Ab trimer in aqueous environment upon D23N mutation, we investigated the D23N 3Ab 11-40 mutant in comparison with the wild type (WT) trimer using extensive replica exchange molecular dynamic (REMD) simulation over 20 000 ns in total. It is noted that the D23N Ab peptides are able to form both parallel and anti-parallel motifs; 38,39 however, in this work, we only considered the D23N 3Ab starting from the parallel structure. Our results indicate that the D23N mutant has higher b-structure and stronger interchain interaction. We also identied seven optimized conformations of solvated D23N 3Ab using the combination of free energy landscape and clustering methods. Finally, molecular docking method was used to evaluate the binding affinity of available inhibitors to D23N and WT Ab 11-40 trimer and monomer in order to reveal the inuence of D23N mutant on AD therapy.
Initial conformation
The three-dimensional bril-like conformation of WT 3Ab peptide was taken from the two-fold 12Ab bril. 40 Then, the residue D23 was mutated to N using the mutagenesis tool in Pymol (Fig. 1). 29 The backbone of trimer was not changed, but the sidechain of D23 was slightly different to N23. The distances between N23 and neighboring atoms remained larger than the corresponding atomic van der Waals (vdW) radii. D23N 3Ab was then inserted into a dodecahedron periodic boundary condition (PBC) box with the volume of $463 nm 3 and solvated with 12 852 water molecules. The mutant trimer was treated with AMBER99SB-ILDN force eld 41 and water molecules were presented utilizing TIP3P water model 42 as described in previous works. 29,43 In addition, the D23N and WT Ab 11-40 monomers were derived from the trimer systems. Both monomers were solvated in a dodecahedron PBC box with size of $270 nm 3 using $8600 water molecules. Na + ions were added to WT Ab 11-40 monomer in order to neutralize the system.
Atomistic simulations
Atomistic simulations were carried out as previously described. 29,44 GROMACS 5.0.6 (ref. 45) was employed to simulate the solvated D23N 3Ab 11-40 system. The non-covalent bond pair cut-off was set at 0.9 nm. The electrostatic potential was treated using the fast smooth particle-mesh Ewald electrostatic method. 46 The vdW interaction was set at 0.9 nm. The rst step of simulation was energy minimization with the steepest descent method, which was followed by a 500 ps positionally restraint simulation in NVT ensemble. REMD simulation was then applied to sample the conformations of solvated D23N (red) and WT 3Ab (blue) in REMD simulation time interval of 300-417 ns at 300 K. The values of WT 3Ab were reproduced with permission of Royal Chemistry Society. 29 3Ab . 48 replicas at various temperatures ranging from 295 to 382 K (ESI †) were simulated as reported in our previous works. 29,43 During REMD simulation, the exchanges were performed every 1 ps. Each replica was simulated over 417 ns. The solvated D23N and WT Ab 11-40 monomers were also simulated for 100 ns at 300 K in order to generate the target for molecular docking. The trimer conformations in REMD simulations were recorded every 10 ps, while monomeric conformations in MD simulations were monitored every 1 ps.
Structural analysis
The Dened Secondary Structure of Protein (DSSP) method was employed to predict the secondary structure term of the D23N 3Ab . 47 The inter-/intra-molecular sidechain (inter-/intra-SC) contacts were counted when the spacing between non-hydrogen atoms are smaller than 0.45 nm. The inter-/intra-molecular hydrogen bond (HB) contacts were counted when the spacing between donor and acceptor is smaller than 0.35 nm and the angle between acceptor-hydrogen-donor is larger than 135 . The collective variable free energy landscape (FEL) was generated with two reaction coordinates, including the C a root-meansquare deviation (RMSD) and gyration of radius R g . The noncovalent bond interaction energy and solvent surface access area (SASA) were calculated using GROMACS tools. 45 The collision cross section (CCS) was computed using the Ion Mobility Projection Approximation Calculation Tool (IMPACT). 48
Binding free energy calculations
The binding free energy of an Ab peptide chain to the other chains within D23N 3Ab was calculated using MM/PBSA method 49-51 as previously described. 29
Molecular docking
The binding conguration and affinity of available inhibitors to Ab trimers and monomers were investigated using Autodock Vina package 52 as described previously. 53 The input le of docking was prepared using AutodockTools 1.5.6. 54 The docking grid was chosen at the center of mass of the peptide with the size of 60 Â 60 Â 60 nm, which is large enough to cover the entire peptides. The exhaustiveness was set as 400.
Results and discussion
REMD simulations of solvated D23N 3Ab REMD simulation is known to be one of the most efficient sampling methods to evaluate the self-aggregation of Ab peptides. 15,16,55,56 Because simulation of the self-aggregation process of Ab peptides is very slow and it is highly unlikely to reach the stable structure of Ab oligomers starting from a random structure, input conformations were obtained from rom bril-like structures, 29,43,44 which was also applied in the case of Ab 11-40 trimer and its D23N mutant. REMD simulations were sufficient as the mean exchange rate of ca. 32% and the Ab conformations entirely walked through the temperature space (Fig. S1 †). The rst 300 ns of REMD simulations were omitted from any analysis due to sidestepping any inclination toward the starting conformation. All computational metrics of the solvated D23N 3Ab were only evaluated over the last 117 ns of REMD simulation at 300 K.
The simulation reached the equilibrium state at 300 K aer 300 ns as indicated by the superposition of secondary structure metrics over various intervals 300-417 and 300-360 ns (Fig. S2 †). All metrics were computed over all the snapshots of the simulation window 300-417 ns. As shown in Fig. 2, the bcontent value diffuses in the range of 11-71% with the mean value of $48 AE 7%. It is signicantly larger than that of WT 3Ab ($42 AE 6%). 29 The shi of b-content distribution under the effect of D23N mutation is clearly shown in Fig. 2. R g uctuates in the range of 1.28-1.64 nm with an averaged value of $1.49 AE 0.05 nm, which is larger than the corresponding value of the WT trimer ($1.43 AE 0.05 nm). 29 SASA of D23N 3Ab ($62.84 AE 2.66 nm 2 ) is slightly smaller than that of WT 3Ab ($63.51 AE 2.59 nm 2 ). 29 The non-covalent bond interaction energy between peptide chains of D23N 3Ab was obtained using the GROMACS tool 4.3, which falls in the range from À515.7 to À160.5 kcal mol À1 (Fig. 2). The average interaction energy is À310.2 AE 53.3 kcal mol À1 , which is signicantly larger than that of WT 3Ab 11-40 (À267.0 AE 67.2 kcal mol À1 ). 29 The stronger interaction energy between establishing chains is observed when the electrostatic repulsion is reduced. The obtained results indicated that the sidechain charge-charge interactions contribute to the instability of the WT trimer. 57 This result implies that D23N 3Ab 11-40 is more stable and forms at higher rate in comparison to the WT trimer.
Effects of D23N mutation on the distribution of secondary structure parameters per residue Fig. 3 shows the distribution of secondary structure terms per residues of the D23N 3Ab including b-, a-, turn-, and coilstructures. Obtained results are consistent with the available solid state NMR data that showed a high neurotoxic Ab 40 oligomer possessing stable N-terminal b-strands. 58 D23N 3Ab adopts parallel U-shape structure consisting of ve domains, which is in good agreement with a recent study. 38 The sequences 11-14, 24-30, and 36-40 mostly form coil structure, while b-structure is dominant in the sequences 15-23 and 31-35. Residues 21-23 in the D23N mutant possess remarkably higher b-structure content compared to those in the WT trimer. Moreover, b-structure content is also observed to increase in other residues, including the sequence 12-20, while it only decreases in several residues, including the sequence 31-34. The loop region (sequence 24-28) forms more coil-structure and less a-/turn-structure in comparison with the WT form. Overall, the signicant decrease in a-structure, an intermediate structure in the Ab folding progress, 59,60 together with the signicant increase in b-structure could speed up the self-aggregation of D23N 3Ab in comparison with that of the WT trimer.
Non-covalent bond contacts
The stability of Ab oligomer depends on the intermolecular non-covalent bonds between constituting chains, involving inter-SC and inter-HB contacts. The distributions of these values are shown in Fig. 4. As mentioned above, D23N Ab 11-40 forms more inter-SC contact (66.0 AE 4.5) than the WT system (61.8 AE 5.2). 29 Similarly, D23N Ab 11-40 adopts more inter-HB contact (12.1 AE 5.0) than WT trimer (10.2 AE 1.7). 29 Moreover, the number of intramolecular contacts of the D23N 3Ab (135.7 AE 7.8) is signicantly smaller than those of WT 3Ab (171.2 AE 8.3). 29 Likewise, the intra-HB of D23N 3Ab 11-40 (6.8 AE 2.0) is lower than that of the WT trimer (7.6 AE 2.9). 29 This result is consistent with the signicant increase in interaction energy between the peptide chains of the trimers obtained using the GROMACS tools described above, as well as with the increase in Gibbs binding free energy between peptide chains derived from MM/PBSA method (vide infra).
Free energy landscape
The collective variable free energy landscape (FEL) of D23N 3Ab was generated using the GROMACS tool named "sham". 45 C a RMSD and R g were chosen as the coordinates as they were successfully used to obtain the FEL for other Ab peptide systems. [61][62][63] The FEL of solvated D23N 3Ab 11-40 is displayed in Fig. 5, which contains four minima centered at (RMSD; R g ) coordinates of (0.48; 1.52), (1.50; 0.61), (0.68; 1.46), (0.66; 1.48). Applying clustering method on the rened MD snapshots located in the free energy holes, we found seven representative conformations of D23N 3Ab . These conformations are noted as A, B, C, D, E, F, and G as shown in Fig. 5. . 4 The distribution of inter/intra-molecular SC and HB contacts of the solvated D23 and WT 3Ab . The results of WT system was reproduced with permission of Royal of Chemistry Society. 29 Fig. 3 Secondary structure terms per residues averaged for all three chains of D23N 3Ab and WT 3Ab . 29 The values of WT 3Ab were reproduced with permission of Royal Chemistry Society. 29 The selected structural parameters of these conformations are provided in Table 1. The dimensions of these conformations, which are indicated by R g , SASA, and CCS, are roughly comparable to those found for the representative structures of the WT trimers. The b-content of these structures is greater than 42%, which is signicantly higher than that found for the representative structures of WT 3Ab, namely MA (37%), MB (40%), and MC (32%). 29 These results are consistent with the analysis of all equilibrated snapshots described above.
The 3-D structures of A-G and their populations are shown in Fig. 6. All conformations exhibit the U-shaped parallel structures. As mentioned above, the b-strand of the N-terminal in the D23N mutant forms more b-structure as previously observed by solid state NMR. 58 It is worthy to note that the central hydrophobic domain is solvent accessible, to which other Ab peptides could dock, resulting in the self-aggregation. 64 Unlike the b-strands in the WT trimer that form crossing structure, 29 the b-strands in D23N 3Ab are parallel to each other. Docking of additional monomer to the parallel structure of D23N 3Ab is much more efficient than that to the crossing structure of the WT trimer. 64 Binding affinity between peptide chains in Ab
trimers
The increase in the interaction energy and non-covalent bond contacts upon D23N mutation of 3Ab 11-40 described above suggest that the peptide chains in the mutant trimer bind more strongly to each other than in the WT trimer. To further access this result, we performed MM/PBSA calculation to evaluate the Gibbs free binding energy (DG bind ) of a peptide chain to the other chains in D23N 3Ab , using the same protocol previously described for WT 3Ab 11-40 . 29 Evaluating binding affinity between peptide chains of the trimer using DG bind obtained with MM/PBSA method is more accurate than using the interaction energy obtained with GROMACS tool. The parameters obtained with MM/PBSA method are provided in Table 2. Replacing the negatively-charged D23 residue by a neutral N residue results in remarkably stronger electrostatic interaction (DE elec ) and slightly stronger vdW interaction (DE vdW ) as a result of weaker repulsion force between the peptide chains. The polar interaction is signicantly stronger in the mutant, which off-set the increase in affinity between the chains. Nevertheless, the magnitude of DG bind increases signicantly (by $22%) when D23 is mutated to N, which is consistent with the increase in the magnitude of interaction energy obtained with GROMACS tool.
Binding of inhibitors to Ab trimers and monomers
In order to gain more insights into how D23N mutation affects the structure of Ab trimers, we compared the binding of twelve experimentally characterized amyloid plaque-binding compounds [65][66][67][68][69][70][71] to D23N 3Ab and WT 3Ab 11-40 using Autodock Vina. 52 This tool has been used to study the binding of ligands to amyloid peptides and provided good correlation Tables 3 and S1 †. Except for one ligand, the estimated binding affinity of the ligands to D23N 3Ab is slightly higher than that to WT 3Ab , however, the difference is negligible. Fig. 7 shows the binding poses of the ligands to the representative conformations of D23N 3Ab (A-G) and WT 3Ab (MA-MC). Overall, the ligands bind to D23N 3Ab in different positions compared to WT 3Ab . The binding poses of the ligands to D23N 3Ab are relatively wide spread, but in most cases they bind to the N-terminal b-strands. While the ligands also bind to the N-terminal domain of WT 3Ab , they mostly dock to the loop regions in similar poses in each conformation. This result suggests that these ligands could inhibit the aggregation of D23N and WT Ab by different mechanisms. Binding of the ligands to the b-strands may inhibit the docking of new Ab chain to the existing oligomers, while binding of the ligand to the loop regions could lead to the destabilization of the oligomers.
In addition, the inhibitors were also docked to the D23N and WT Ab 11-40 the representative structures of the monomers obtained from FEL and clustering methods (Fig. S4 †). D23N mutation cause signicant changes in the structure of Ab 11-40 monomer (Fig. S4 †). The binding poses of the inhibitors to D23N monomer are different from those to WT Ab 11-40 monomer (Fig. S5 †). The average ligand binding affinity of D23N monomer (À5.0 AE 0.2 kcal mol À1 ) is 0.7 kcal mol À1 smaller than that of D23N trimer (À5.7 AE 0.3 kcal mol À1 ) (Table S2 †). Similarly, the average ligand binding affinity of WT Ab 11-40 (À4.8 AE 0.2) is 0.7 kcal mol À1 smaller than that of WT 3Ab 11-40 (À5.5 AE Fig. 7 Binding poses of twelve ligands to the representative conformations of D23N 3Ab (A-G) (this work) and WT 3Ab (MA-MC) (obtained from our previous study) 29 generated using Autodock Vina protocol. 52 The residues highlighted by red color form non-bonded contacts to the ligands. Spheres denote the C a atoms of the N-terminal. Table 3 Binding affinity (kcal mol À1 ) of selected inhibitors to D23N and WT 3Ab No.
PubChem ID Ref.
D23N 3Ab WT 3Ab 11 0.3 kcal mol À1 ). This result indicates that the inhibitors have higher affinity for the trimers than for the monomers. It also shows that D23N mutation also causes changes in ligand binding poses of the monomers, further corroborating the results obtained for the trimers.
Conclusions
Atomistic structural and dynamic information of the truncated Iowa Ab mutant trimer (D23N 3Ab ) were extensively simulated using REMD with 48 replicas over simulation time of 417 ns per replica ($20 000 ns of total simulations time).
Replacing the negatively-charged D23 residue by the neutral residue N increases b-structure content from $42% to $48%, which occurs concomitantly with a shi in the hydrophobic bstrand core from crossing to parallel arrangement (Fig. 7). Subsequent docking of other Ab chains via dock-lock mechanism 64,74 to the existing oligomer with parallel hydrophobic core could occur much more efficiently than to the oligomer with crossing core. The number of non-covalent contacts increases sizably in the D23N mutant trimer, which is consistent with the increase in the magnitude of both the interaction energy (from À267.0 AE 67.2 kcal mol À1 to À310.2 AE 53.3 kcal mol À1 ) derived with the GROMACS tool and the Gibbs free binding energy (from À35.82 AE 7.89 to À43.67 AE 15.90) obtained with MM/ PBSA method. Altogether, our results show that D23N mutation induces signicant changes in structure and dynamics of 3Ab 11-40 that allow the mutant peptide to aggregate at faster rate, consistent with previous experimental results on the formation of Iowa Ab brils. 30 Molecular docking shows that D23N mutation causes signicant changes of Ab 11-40 structure. These changes lead to differences in ligand binding poses of D23N and WT trimers despite having similar binding affinity. The ligands mostly bind to the N-terminal b-strand of the D23N mutant trimer, while they prefer to bind to the N-terminal loop region of the WT trimer. Likewise, these compounds bind to D23N and WT Ab 11-40 monomers at different binding positions while having the same range in binding affinity. This result provides important information for further study in drug design for different types of AD. Rigorous MD simulations are needed to gain further insights into this aspects.
Conflicts of interest
There are no conicts to declare. | 2019-04-03T13:08:45.381Z | 2018-12-12T00:00:00.000 | {
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29451734 | pes2o/s2orc | v3-fos-license | Antigenic relationships of murine coronaviruses: Analysis using monoclonal antibodies to JHM (MHV-4) virus
Monoclonal antibodies were produced to JHMV-DL, a neurotropic member of the mouse hepatitis virus (MHV) or murine coronavirus group. Of 23 antibodies isolated, 10 were specific for the major envelope glycoprotein, gp180/90, 10 for the nucleocapsid protein, pp60, and 3 for the minor envelope glycoprotein, gp25. Eleven different MHV isolates were used in antibody binding assays to study antigenic relationships among the viruses. Each MHV isolate tested had a unique pattern of antibody binding, indicating that each is a distinct strain. Conservation of JHMV-DL antigenic determinants varied among the three proteins, with pp60 showing intermediate conservation, gp180/90 little conservation, and gp25 marked conservation in the different MHV strains. Monoclonal antibodies to pp60 proved most useful in delineating antigenic relationships among MHV strains. These antigenic groups correlated with pathogenic types, indicating that pp60 may be one of the gene products which mediates the distinct disease patterns manifested by different murine coronaviruses.
INTRODUCTION
Coronaviruses are enveloped viruses containing a single-stranded RNA genome of positive polarity Sturman and Holmes, 1983) . The murine coronavirus or mouse hepatitis virus (MHV) group is of particular interest because of the wide range of diseases produced by its members . Although the majority of MHV strains usually cause inapparent or latent gastrointestinal infections in nature (Gledhill and Niven, 1955 ;Rowe et at, 1963), different MHVs have been reported to spontaneously or experimentally produce hepatitis, enteritis, peritonitis, lower respiratory infections, panencephalitis, demyelination, choroi-' To whom correspondence and reprint requests should he addressed .
Despite marked variations in pathogenicity, the MHVs have relatively simple structural features, usually consisting of a major envelope glycoprotein, a minor envelope glycoprotein, and a nucleocapsid protein . The major glycoprotein, gp180/90, forms the projecting spikes or peplomers . These are important for cell attachment (Sturman and Holmes, 1983) and may influence tropism for specific tissues . The minor glycoprotein, gp25, is largely an internal protein, although a small segment projects through the virion envelope (Sturman, 1982) . This protein is probably analogous to the matrix protein of other enveloped viruses and is thought to play a role in the control of viral maturation and establishment of virion stability (Holmes et at, 1982) . The nucleocapsid protein, ppGO, is intimately associated with the viral RNA . The simplicity of the struc-ture of the virion makes the MHV group an ideal subject for comparative studies of pathogenesis, as, in principle, variations in disease patterns can be correlated with changes in protein structure.
The relatedness of the MHV strains has been examined by a number of methods, including cross-neutralization with hyperimmune serum (Hierholzer, 1979 ;Wege, et at, 1981) and, more recently, kinetic neutralization (Childs et at, 1983). Most MHV strains are antigenically distinct by these methods, although the MHV3 and A59 strains are very similar. Analysis of the viral polypeptides has shown that some MHVs have proteins with unique migration patterns by sodium dodecyl sulfate polyacrylamide gel electrophoresis Cheley et at, 1981; . More recently, several studies have compared the oligonueleotide maps of closely related MHVs that cause different diseases in order to identify the genes responsible for different disease patterns Wege et al., 1981 ;Stohlman et at ., 1982) .
JHM virus (JHMV) is a neurotropic MHV which may cause encephalitis and demyelination (Bailey et at, 1949;Weiner, 1973) . Its structural features (Massalski et al., 1982), proteins and genome , are those of a typical MHV . In animal models of chronic demyelination, JHMV persistence has been demonstrated for extended periods (Stohlman and Weiner, 1981;. Recently two plaque-size variants of JHMV have been isolated and characterized : a large plaque isolate, JHMV-DL, with a high propensity to acute encephalitis, and a smallplaque variant, JHMV-DS, which predominantly causes chronic demyelination (Stohlman et at, 1982) . In this manuscript, we report the use of a panel of JHMV-DLspecific monoclonal antibodies to study the antigenic relationships of the structural proteins of 11 separate MHV isolates .
MATERIALS AND METHODS
Viruses and cells. Viruses were propagated in DBT cells as described previously (Stohlman and Weiner, 1978), except when MURINE CORONAVIRUS ANTIGENS 297 virus in serum-free media was desired for use in radioimmunoassay (RI A) . In this case, infected cells were first cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco Laboratories, Grand Island, N. Y at -70°. The derivation and characterization of the two plaque morphology variants of JHMV (MIIV-4), DL (large plaque) and DS (small plaque), have been described recently (Stohlman et at, 1982) . MHV-1, MHV-2, MHV-3, and MHV-S viruses were plaque-purified from virus stocks obtained from Dr . M. Collins, Microbiological Associates, Bethesda, Maryland and K . Fujiwara, University of Tokyo, Tokyo, Japan . The MHV-K, MHV-D, and MHV-Nuu viruses were obtained as cloned stocks from Dr . K. Fujiwara . The MHV-M virus, an isolate from nude mice with wasting disease, was obtained from Dr . M. Collins . Vesicular stomatitis virus (VSV) and herpes simplex virus, type I (HSV-1), were obtained from Dr . P . Brayton and Dr. D . Willey, respectively, both of the University of Southern California, School of Medicine .
For the production of the monoclonal antibodies, M5 cells, a horse serum-adapted line of SP2/0-Ag .14, obtained from Dr . J . Davie, Washington University, St . Louis, Missouri, were grown in DMEM supplem e n t e d w i t h 1 0 % h o r s e s e r u m , 20 mM HEPES, 100 IU/ml penicillin, 100 pg/ml streptomycin, 2 mM L-glutamine, 10 mM MEM nonessential amino acids (Gibco Laboratories, Grand Island, N . Y.), and 5 X 10_ 6 M 2-mereaptoethanol . During selection, HAT was added to the medium (final concentrations : 1 X 10_ , Mhypoxanthine, 4X10-' M aminopterin, and 1 .6 x 10-5 M thymidine) (Littlefield, 1964). Virus neutralization. The ability of the monoclonal antibodies to neutralize virus was assayed as previously described (Stohlman and Weiner, 1981) . Briefly, 25-µl serial dilutions of antibody (heat inactivated at 56° for 30 min) were added to wells of a 96-well microtiter plate ; subsequently, 25 µl of a virus suspension containing 10-TCID6" d o s e s ( 5 0 % t i s s u e c u l t u r e infective doses) was added to each well . The plates were incubated at 37° for 1 hr, and then 2 X 10°DBT cells were added to each well . The neutralizing titer was expressed as the highest dilution of antibody w h i c h p r e v e n t e d C P E i n 5 0 % o f t h e w e l l s . Antibody binding assay. The solid phase radioimmunoassay (RIA) for antiviral antibodies has been described previously (Fleming et al, 1983) . Viral antigens were used in excess; preliminary assays showed that each virus stock contained sufficient antigen to produce maximal binding with a hyperimmune anti-MHV standard serum. After cloning, 25 µl of supernatant from established hybridomas was assayed at 10 -1 dilution. Serial dilutions of antibody showed that this concentration fell within the linear region of the assay . In'eross reactive RIA, the binding of antibodies at 10-1 dilution was tested against homologous virus (JHMV-DL) and compared to the binding to heterologous viruses .
Relative binding was expressed by a modification of the convention of Gerhard et at (1981). The counts per minute (cpm) bound to homologous (JHMV-DL) antigen w e r e n o r m a l i z e d t o 1 0 0 % . Binding of g r e a t e r t h a n 5 0 % o f t h i s v a l u e w a s c o nsidered strongly positive; 2 5 -5 0 % , m o d e rately positive ; l e s s t h a n 2 5 % b u t m o r e t h a n twice background, weakly positive; and less than twice background, negative . Results shown in the block diagrams are the composite of at least three assays, each of which consisted of quadruplicate samples .
Antisera. Hyperimmune antisera to JHMV-DL and A59 were made by serial immunizations of C57BL/6J (B6) mice obtained from Jackson Laboratories, Bar Harbor, Maine. Normal mouse serum free of anti-MHV antibody was obtained from B6 mice immediately after being received . Control antibodies used included monoclonal anti-mouse Ia (Harmon et al., 1982), and rabbit anti-HSV-1, kindly supplied by Dr . D . Willey of the University of Southern California, School of Medicine. FLEMING ET AL.
Preparation of monoclonal antibodies. The technique of Kohler and Milstein (1975) as modified by Harmon et at, (1982), was followed. B6 mice, 6 weeks of age, were inoculated intraperitoneally with approximately 105 plaque-forming units of JHMV-DL grown in serum-free medium . Secondary immunization, usually 6 weeks later by the intravenous route, also consisted of 105 plaque-forming units of virus in serumfree medium . Suspensions of immune spleen cells were fused to M5 cells at a ratio of 2.5: 1 u s i n g 3 4 % p o l y e t h y l e n e g l y c o l MW 1500 (Aldrich Chemical Co ., Milwaukee, Wis.) at 37°. The suspension was then washed, resuspended in HAT medium, and cultured in 24-well plates at 1 X 106 cells per well, supplemented by 1 X 10 6 feeder spleen cells/well prepared from nonimmune B6 mice . Following incubation for 4 days at 37°, half of the medium was removed from each well and replaced with fresh HAT medium containing 1 X 106 feeder cells . On Day 8 this step was repeated with HT media (aminopterin-free) and feeder cells. On Day 11, wells were screened by RIA for antiviral antibody . Cells in positive wells were cloned by visual inspection and limiting dilution in 96-well plates. Clones were expanded, and supernatants were subsequently harvested and reassayed by RIA . Monoclonal antibodies were assayed for immunoglobin class and subclass by Ouchterlony immunodiffusion, using antiserum to murine immunoglobulin isotypes (Litton Bionetics, Kensington, Md .) .
Radioirnmunopreeipitation . The specificity of the monoclonal antibodies for viral proteins was determined by radioimmunoprecipitation (RIP) . DBT cells were inoculated with virus at a multiplicity of infection of 1-5 for 1 hr at 37°. After removal of the inoculum, the cultures were incubated in the presence of serum-free DMEM containing 1 jig/ml actinomycin D . The medium was removed and replaced with prewarmed methionine-free DMEM (MFDMEM) for 15 min . The medium was again removed and replaced with prewarmed MFDMEM containing 20,uCi/ml°5 S-methionine (New England Nuclear, Boston, Mass.) . Following incubation for 30-45 min, the cultures were placed on crushed ice, washed 2 times with ice-cold PBS, and solubilized with a buffer composed of 10 mM Tris-HCL pH 7 .4, 150 mM NaCl, 0 . 5 % N P -4 0 , 1 m g / m l N -p -t o s y l -Llysine-chlormethyl ketone HCL (TLCK), 200µg/ml phenylmethylsulphonyl fluoride (PMSF), and 500 u/ml aprotinin . The lysate was clarified by centrifugation at 500 g for 5 min and stored at -70°. For immunoprecipitation, the lysates were adjusted to 0. 2 % s o d i u m d o d e c y l s u l f a t e ( S D S ) a n d i ncubated with an equal volume of polyvalent antiserum or monoclonal antibody as described by McMillan et al. (1981) . The immune complexes were adsorbed to a suspension of protein-A bearing Staphylococcus aureus as described by Kessler (1981) . The final pellet was resuspended in a buffer containing 67 mMTris-HCL, pH 6 .8,2 . 6 6 % SDS, 5 . 0 % 2 -m e r c a p t o e t h a n o l , h e a t e d a t 56° for 2 min, and then centrifuged for 1 .5 min at 10,000 g. The supernatants were ana l y z e d b y e l e c t r o p h o r e s i s o n 6 -1 5 % d i scontinous slab polyacrylamide gels .
RESULTS
Production and characterization of monoclonat antibodies to JHM-DL virus Twenty-three cloned hybridomas produced antiviral antibodies as determined by RIA . The specificity of each antibody was determined by precipitation of JHMV-DL proteins from a lysate of infected cells . Figure 1 shows examples of the immunop r e c i p i t a t e s a n a l y z e d o n s i x 1 5 % g r a d i e n t polyacrylamide gels . Lysates harvested late in infection contained various quantities of the degradation products of the nucleocapsid protein (lanes 7, 8, 9, 10, and 11 in Fig. 1 and lanes 6 and 8 in Fig. 5) . In addition, a few monoclonal antibodies immunoprecipitated other weak protein bands (see lane 13 in Fig . 1 and lane 11 in Fig. 5) which could be eliminated by increasing the SDS to 0. 5 % i n t h e r e a c t i o n mixture . Based on the data obtained by immunoprecipitation, the 23 monoclonal antibodies were placed in three groups . Table 1 shows. that 10 antibodies reacted with the major glycoprotein, gpISO/90, 10 reacted with the nucleocapsid protein, pp6O, and 3 reacted with the minor envelope glycoprotein gp25 . All the antibodies were tested for their ability to neutralize JHMV-DL . Six of the ten that reacted with gp18O/90 neutralized JHMV-DL . None of the antibodies reactive with either pp6O or gp25 were able to neutralize JHMV-DL (Table 1) . The heavy-chain immunoglobulin isotype of each antibody was tested by immunodiffusion . All the antibodies reacted with only a single anti-isotype antiserum. The isotypes for each monoclonal antibody are shown in Table 1 .
Reactivities of antibodies with nucleocapsid protein pp60. Ten monoclonal antibodies reactive with the nucleocapsid protein of JHMV-DL, pp6O, were used to examine the antigenic conservation of this protein in MIIV strains that induce diverse types of disease. Antigenic preservation was determined in an RIA which tested the 10 antibodies with different MHV strains serving as antigens . Table 2 shows an example of this type of experiment .
Anti-pp60 antibodies were tested for their ability to bind to the homologous antigen (JHMV-DL), the small-plaque variant of this virus (JHMV-DS), and a related murine coronavirus (MHV-A59), as well as control antigens (HSV-1, VSV) . Table 2 s h o w s b i n d i n g o f a t l e a s t 2 5 % o f t h e v a l u e s for homologous virus in 6 of 10 reactions of anti-pp60 antibodies with MHV-A59 and none of 10 with JHMV-DS . Since viral antigens were used in excess and binding of antibodies to the glycoproteins of JHMV-DS was equal to the binding to the glycoproteins of homologous virus (see below), we do not attribute the very low binding of antibodies to JHMV-DS nucleocapsid protein, pp6O, to limited antigenic density of the viral preparation. Anti-pp6O monoclonal antibodies did not react with control viral antigens, and control monoclonal antibody did not react with MHV antigens . The relative binding of antibodies to different MHV strains in a larger series of experiments was used to construct the block diagram presented in Fig . 2. The pattern of reactivities indicates a moderate degree of antigenic conservation of ppGO among MHV strains . By inspection, the viruses tested could be divided into two broad groups based on their reactivities . The first group consisted of A59, MHV-3, MHV-D, MHV-K, and MHV-Nuu viruses, all of which did not react with monoclonal antibodies J.1.1, J .3.14, J.3.7, and J .3.15 . The antigenic sites recognized by these antibodies are apparently conserved in the second group, composed of MHV-2, MHV-S, and MHV-1 viruses . In addition to these two groups, two individual viruses, JHMV-DS and MHV-M, were notable for their l a c k o f m a r k e d b i n d i n g ( l e s s t h a n 2 5 % r p m versus homologous virus) with any of the 10 anti-pp60 monoclonal antibodies tested . Reactivities of antibodies with major glycoprotein 180/90. The major virion envelope glycoprotein, gplSO/90, is capable of a number of functions, including recognition of the host cell receptor (Sturman and Holmes, 1983). It was therefore of interest to investigate the antigenic relationships of gp18O/90 in a number of different MHV strains which produce a variety of diseases. All monoclonal antibodies recognized antigenic determinants of the small plaque variant JHMV-DS equally well as the immunogen, JHMV-DL, indicating the close antigenic relationship of 'Monoclonal antibodies were obtained from hybridoma tissue culture supernatants. Antigen specificity was determined by RIP, neutralizing titer by microassay, and immunoglobulin isotype by immunodiffusion as described under Materials and Methods .° Symbol (-) indicates no neutralization at 1 :4 dilution . these two viruses (Fig . 3) . By contrast, very few of the antigenic determinants on the other MHVs were closely related to those of JHMV-DL, indicating the lack of conservation of JHMV major glycoprotein antigens among the rest of the MHV strains .
Several antibodies, such as J.1.2, J .7.18, J.2.2, and J .7.2, recognize only JHMV variants DL and DS and thus may appear to have identical specificities. Nevertheless, differences in fine specificity were demonstrated in further experiments . We first isolated variant JHM viruses which escaped neutralization by monoclonal antibodies . When these variant viruses were used as antigens, each of the 10 anti-gp180/ 90 monoclonal antibodies in our panel showed a distinctive pattern of reactivity to them, indicating each antibody in this set recognized a unique antigenic determinant (Fleming, unpublished observations).
Reactivity of antibodies with minor glycoprotein gp25. The smaller viral glycoprotein, gp25, is embedded within and extends through the viral envelope . It has been suggested that it may function as a matrix protein and is essential in viral budding and in the formation of the viral envelope (Holmes et aL, 1982) . Three monoclonal antibodies were obtained which are specific for gp25 . This protein appears to be highly conserved among the MHV strains (Fig . 4) . Nevertheless, this finding must be regarded as tentative, in view of the small numbers of available antibodies with specificity for gp25 .
Cross immunoprecipitations. To confirm the pattern of antibody binding determined by RIA, anti-JHMV-DL monoclonal antibodies were tested for their ability to im munoprecipitate the appropriate viral protein from a lysate of cells infected with the related MHV, A59. Figure 5 shows that those anti-JHMV-DL antibodies (J.7.5, J.2.1, J .1 .3, and J .2.7) which react with A59 by RIA were also able to immunoprecipitate the appropriate A59-specified protein, indicating that the shared antigens detected by RIA were in fact on the analogous protein . By contrast, those antibodies that were unreactive with A59 virus by RIA (J.2.5, J1.2, J .3.7, and J.1.1) did not immunoprecipitate detectable radiolabeled A59 virus proteins . Taken together, the immunoprecipitation data confirm the specificities of the anti-JHMV-DL antibodies as determined by RIA .
DISCUSSION
Although marine coronaviruses have a relatively simple structure, they cause a wide variety of diseases . In an attempt to understand the basis for their diverse pathogenicities and to obtain information on the antigenic characteristics of these viruses, we have used 23 monoclonal an- IgG 2b tibodies derived from the immunization of mice with the neurotropic strain JHMV-DL to examine the relationships of the structural proteins of the principal MHV strains . Assessment by RIA and RIP showed that the degree of conservation of JHMV-DL antigens, as judged by this panel of antibodies, varied among the three principal MHV structural proteins. The nucleocapsid protein pp60 showed intermediate conservation, the major glycoprotein gp180/90 little conservation, and the minor glycoprotein gp25 strong conservation . Each MHV strain had a unique pattern of reactivities to the set of monoclonal antibodies used, confirming previous studies by Childs et at (1983) which showed that these MHV isolates are in fact separate and distinct strains.
Relative binding to anti-pp60 antibodies allowed grouping of the MHV strains into several antigenic families . In general, this group of antibodies showed substantial binding to all the murine coronaviruses tested, with the exception of JHMV-DS and MHV-M, indicating that the nucleocapsid protein antigens are substantially conserved in most MHV strains . Analysis of the relatedness of the pp6o proteins divided 'Sample RIA: Data are given as counts per minute with mean value and one standard deviation of four replicate samples. Cpm for JHMV-DS and A59 are also expressed as a percentage of cpm for JHMV-DL (parenthesis) . Symbol (-) indicates background epm and NT indicates that the interaction was not tested .
All antibodies were tested at 10-' dilution. Control antibodies are anti-mouse la (monoclonal antibody produced by an identical protocol) and anti-HSV-1 (hyperimmune rabbit serum) . the viruses into two groupings. The first consisted of A59, MHV-3, MHV-D, MHV-K, and MHV-Nuu viruses, which were onreactive with monoclonal antibodies J .1.1, J.3.14, J.3.7, and J .3.15. The second group, consisting of MHV-2, MHV-S, and MHV-1 viruses, conserved these antigenic specificities, as evidenced by strong reactions with the same set of monoclonal antibodies . Although these strains have not all been previously compared in a single study, previous investigations do support this general division . For example, the oligonucleotide maps of MHV-1 and MHV-S are distinct from the other MHVs examined , as are the peptide maps of the pp6O from these two strains (Cheley et at, 1981) . In addition, the oligonucleotide maps of A59, MHV-3, MHV-D, and MHV-K are very similar Lai, unpublished data) . MHV-M has an oligonucleotide map which is very different from any other MHV examined (Lai, unpublished data), and it is therefore not surprising that there is little recognition of its antigens .
The finding that none of the 10 anti-pp6O antibodies showed moderate or strong binding to JHMV-DS was very unexpected, since JHMV-DL and JHMV-DS have a 302 FLEMING ET AL . common passage history, and their genomes share all but two oligonucleotide spots (Stohlman et aL, 1982). Recently, oligonucleotide fingerprints of mRNA No . 7, which encodes pp60, were studied in JHMV-DL and JHMV-DS. The fingerprints of the two variants were indistinguishable (Lai, unpublished observations) . We presently have no firm explanation for the difference between the data obtained with monoclonal antibodies and oligonucleotide fingerprinting . Very likely, the monoclonal antibodies detected antigenic sites which are represented by genetic sequences not detectable by T 1 -oligonucleotide fingerprinting . This striking result implies that there is a significant antigenic change in the nucleocapsid protein of the small plaque variant, JHMV-DS . The lack of antigenic differences among JHMV-DL and JHMV-DS in the other two virion proteins suggests that the change in pp60 may be correlated with differences in pathogenicity between the two viruses (Stohlman, et at, 1982) . However, this conclusion is provisional and should be supported by further studies, such as the analysis of nonstructural proteins and the complete sequencing of the genomes of the viruses . Gp180/90 is an external protein which serves as the target for neutralizing antibody, induces cell fusion, and may play a key role in determining host cell range (Holmes et at, 1982;Collins et at, 1982) .
None of the anti-gp180/90 monoclonals indicated any major antigenic conservation of this protein among the wide range of murine coronaviruses, except for JHMV-DS . The fact that none of the gp180/90 FLEMING ET AL . FIG. 4. Reactivities of monoclonal antibodies with the MHV minor glycoprotein, gp25 . The binding of anti-gp25 monoclonal antibodies in RIA to different MHV antigens is expressed as described for Fig. 2 . antigens of the other MHVs shared many determinants with JHMV is not surprising, as marked variability of external proteins, usually in the context of conservation of internal proteins, has been well-documented in viruses such as vesicular stomatitis virus (Doel and Brown, 1978), poliovirus (Nottay et aL, 1981), and the murine retroviruses (Niman and Elder, 1982) . This phenomenon may relate to the high mutation frequency of RNA viral genomes in general and the selective pressure that host immune systems may preferentially exert on neutralization-reactive surface components of viruses (Holland et at, 1982) .
Antigenic determinants of the minor glycoprotein gp25 were highly conserved among the MHV strains tested. This result was not unexpected, since gp25 is thought to serve as a matrix protein (Holmes et aL, 1982 ;Sturman, 1982), and therefore it is possible that many mutations involving gp25 might be lethal to the virus . This finding is consistent with .previous studies showing that the matrix proteins of vesicular stomatitis virus (Doel and Brown, 1978) and influenza virus (Laver and Downie, 1976) are highly conserved among different strains .
Studies of viral pathogenicity are also consistent with the divisions proposed based on the reactivity with the anti-pp60 monoclonal antibodies . Most MHV strains cause hepatitis under natural or appropriate experimental conditions . Nevertheless, the members of the first group characteristically produce other diseases in addition to hepatitis: A59 can cause demyelination choroidoependymitis (Virelizier et at, 1975) MHV-D enteritis (Ishida et at, 1978a), and MHV-K myeloproliferation in nude mice (Ishida et at,197Sb) . By contrast, the members of the second group (MHV-2, MHV-S, and MHV-1) have been reported to produce primarily hepatitis ; MHV-1 and MHV-S have also been considered to be of relatively low pathogenicity (Cheley et at, 1981;. Thus using the present panel of monoclonal antibodies to JHMV-DL, antibodies to pp60, rather than gp180/90 or gp25, are most useful in establishing antigenic,relationships both among MHV strains and between plaque variants of one MHV strain, JHMV . The fact that these antigenic divisions to some extent correlate with pathogenicity raises the possibility that pp60 may play an important role in disease potential . However, genetic regions coding for other gene products may contain alterations which would result in variations not detectable in this study, as shown pre-viously by oligonucleotide fingerprinting of -HMV-DL and JHMV-DS (Stohlman et at, 1982) . Therefore, these conclusions should be considered tentative . Also, the major glycoprotein, gp180/90,is highly variable and may be expected to influence viral pathogenicity, e.g., by controlling tropism for specific cell types . This has already been suggested in one study in which relative hepatotropism among MHV strains may be correlated with changes in the gene for gp180/90 . Taken together, these findings imply that determinants on both gp180/90 and pp60 of MHV may play critical differential roles in pathogenesis. Such a conclusion would be consistent with the detailed information available concerning the genetic and molecular bases of pathogenicity of influenza (Rott, 1979) and reoviruses (Fields and Greene, 1982). For these viruses, virulence has been shown to be multigenic, although individual genes are responsible for different aspects of pathogenesis. Further studies, including an evaluation of the role of nonstructural proteins, will be needed to extend these conclusions to the murine coronaviruses . | 2018-04-03T01:25:37.223Z | 1983-12-01T00:00:00.000 | {
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18356930 | pes2o/s2orc | v3-fos-license | Advancing the Field: Evidence for New Management Strategies in Invasive Fungal Infections
Invasive fungal infections (IFI) are a significant cause of morbidity and mortality in the immunocompromised. The traditional diagnostic methods of culture and histological examination lack sensitivity and often only make a diagnosis late when the fungal burden is high, reducing the chances of cure even with the availability of new more potent and less toxic antifungal agents. New non-culture-based serological and PCR assays have been developed. These appear more sensitive and are able to make an earlier diagnosis as compared with traditional diagnostic methods. Early diagnosis is central to reducing IFI-related morbidity and mortality. This review describes the diagnostic potential of the new serological and PCR assays and outlines how these assays have been incorporated into algorithms to improve the management of IFI.
Introduction
Invasive fungal infections (IFI) remain as a major cause of mortality in those undergoing hematopoietic stem cell or solid organ transplantation, chemotherapy for hematological malignancies, patients in intensive care units (ICU) and those who are HIV infected. Candida is now the fourth most common cause of bloodstream infections and invasive candidiasis (IC) has been associated with an attributable mortality of 49 % in some ICUs in the United States of America (USA) [1,2]. More recently, mortality rates of up to 60 % have been reported with invasive Aspergillus infections in high-risk hematology patients [3, 4•]. The numbers that die from cryptococcal meningitis (CM) is estimated at 625,000 with 80 % (500,000) of these deaths occurring in sub-Saharan Africa [5•].
The economic costs of IFI are also substantial. In the USA the mean total hospital costs of invasive aspergillosis (IA) have been estimated at US$96,310 [6]. A study performed in one Australian center reported that treatment of IFI in allogeneic hematopoietic stem cell transplantation (HSCT) recipients and patients with acute myeloid leukemia was estimated at AU$30,957/case, increasing to AU$80,291/case if admitted to ICU, as compared to matched controls [7].
These high mortality rates and economic costs occur despite the availability of antifungal agents with improved potency and less toxicity. Early treatment has been related to improved outcomes but this is dependent on diagnostic tests that are both rapid and highly sensitive [8]. As traditional culture-and histology-based diagnostic methods have poor sensitivity and often only make a diagnosis late when the fungal burden is high, research has turned its focus in the last two decades to the development of serological and molecular assays with improved accuracy and rapidity of diagnosis [9]. This review will focus on the diagnostic capabilities of new serological and molecular assays and how these assays are incorporated into screening and pre-emptive (or as more accurately renamed diagnostic-driven (DD)) strategies for improved management of IFI.
Serological Markers for the Diagnosis of IFI
The BDG, a polysaccharide cell wall component of many fungi including Candida, Aspergillus and Fusarium, is released during invasive infection. Four commercial tests have been developed for the detection of BDG including Fungitell (Associates of Cape Cod, East Falmouth, MA, USA), Fungitec-G (Seikagaku Cooperation, Tokyo, Japan), Wako-WB003 assay (Wako Pure Chemical Industries, Osaka, Japan) and Maruha (Maruha-Nichizo Foods Inc, Tokyo, Japan). These kits vary according to cut-off value used for a positive result and the detection method (colorimetric or turbimetric). The BDG is detected in a patient's serum by incubating serum with lyophilized horseshoe crab coagulation factor. If BDG is present, then the coagulation cascade is initiated.
The BDG assay has been extensively examined for its diagnostic performance in patients undergoing chemotherapy for hematological malignancies. A recent meta-analysis reported variable sensitivity and specificity (50 %-8 5 % and 80 %-99 %, respectively) [10••]. This variability is due to the heterogeneity of study designs and the populations included. The BDG has also been shown to make an earlier diagnosis of IFI as compared with culture by a median of 5-10 days [11,12]. As a result, BDG assay has been included as an indirect microbiological criterion in the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) definitions of IFI and has been recommended for use in clinical trials and routine clinical practice by the European Conference on Infections in Leukemia (ECIL) guidelines [13, 14•].
The BDG has been less extensively examined in ICU patients. Posteraro et al., examined the diagnostic performance of the assay for IC in critically-ill ICU patients and compared it to the Candida score and colonization index of Leon and Pittet, respectively [15••, 16, 17]. The diagnostic accuracy was found to be greater for a single positive BDG value of >80 pg/ml (cut-off of Fungitell assay) as compared with the Candida score and colonization index (receiver operating characteristic (ROC) area under the curve (AUC) 0.98, 0.80 and 0.63, respectively). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were 93.7 %, 83.6 %, 75 % and 98.6 %, respectively. In addition, BDG was detected 1-3 days prior to blood culture detection.
The evaluation of the BDG assay in bronchoalveolar lavage (BAL) fluid has been very limited, especially in comparison to galactomannan and PCR. The PPV was low (20.0 %) for 109 immunocompromised cases of proven, probable or possible IFI detected using the EORTC/MSG definitions [18]. In comparison to Candida PCR, the Fungitell BDG assay has been reported as having lower sensitivity for IC (80% vs. 56%; p=0.03), with similar specificity (70 % vs. 73 %; p=0.31) [19••].
Glucan is ubiquitous in the environment so false positivity can occur in patients undergoing hemodialysis or given blood products using cellulose materials, surgical patients in contact with cotton gauze, receiving β-lactam antibiotics and with bacteremia. Levels decline with antifungal therapy and a reduction in BDG levels with antifungal therapy has been associated with improved survival. However, the assay is not available in many institutions and is costly. Based on studies to date, the current role of the BDG assay is as an adjunct microbiological test for the diagnosis of IC.
More recently, BDG has been used to diagnose Pneumocystis jirovecii pneumonia (PJP). BDG is a component of the cyst wall of P. jirovecii. In a bivariate meta-analysis of 14 studies that included 357 cases of PJP and 1723 controls without PJP, the average sensitivity and specificity were determined as 94.8% and 86.3%, respectively, with positive and negative likelihood ratios (LR) of 6.9 and 0.06, respectively [20]. The area under the hierarchical summary ROC curve was 0.97. This study indicates that BDG may have potential for the diagnosis of PJP but further clinical evaluation is required.
Candida Mannan Assays
Mannan is a polysaccharide cell wall component of Candida and is released into blood during IC. Candida mannan antigen and antimannan antibody assays (Platelia Candida Antigen and Antibody assays) are commercially available from Bio-Rad (Marnes-la-Coquette, France) in Europe only. The cutoffs for test positivity are defined as 0.5 ng/mL for mannan and 10 arbitrary units/mL for antimannan antibody. Values of 0.25-0.5 ng/mL and 5-10 arbitrary units/mL are considered as indeterminate. Use of both tests in parallel improves sensitivity. A review published by ECIL showed that the sensitivity of the mannan antigen and antimannan antibody assays when used individually were 58 % and 59 %, respectively, but when used together, the sensitivity increased to 83 % with no significant reduction in specificity [21]. Based on these data it is now recommended that the tests are used in combination to diagnose IC. In addition, the assays performance varies according to Candida species. It performs best for C. albicans (sensitivity of 80-100 %), followed by C. glabrata and C. tropicalis, and it performs least well for C. parapsilosis and C. krusei (sensitivity of 40-50 %).
Bio-Rad has developed new detection assays, namely, Platelia Candida Ag Plus (Ag-Plus) and the Platelia Candida Ab Plus (Ab-Plus) which have been evaluated using a collection of stored serum samples obtained from 21 patients with microbiologically proven IC and 30 controls [22•]. All patients included in this study were undergoing myeloablative chemotherapy for hematological malignancies. The sensitivity and time to detection of IC were not significantly better than for the conventional Platelia Candida assays and specificity was reduced by 50 %. The reduction in specificity was determined by logistic regression analysis to be due to the detection of cases of superficial candidiasis by the Ag-Plus assay.
In a retrospective study of 45 patients either the Platelia Candida Antigen and Antibody assays were positive a minimum of two days prior to blood cultures. In addition, the performance depends on the patient population and the phase of treatment. Non-neutropenic surgical patients are more likely to have positive antibodies firstly but neutropenic populations undergoing chemotherapy are more likely to present with antigenemia [23]. Overall, further evaluation of the precise role of Candida mannan antigen and antimannan antibody in different populations is required before these assays are adopted into clinical practice for use in diagnosing IC.
Galactomannan Assay
Galactomannan (GM) is a heat-stable polysaccharide present in the cell wall of most Aspergillus species. It is also present in the cell wall of some Penicillium species. During invasive infection it is released from growing hyphae and can be detected in bodily fluids including serum, BAL and cerebrospinal fluid (CSF). Bio-Rad has developed a commercially available assay, the Platelia Aspergillus GM enzyme-immunosorbent assay (EIA). The original cut-off was an optical density index (ODI) of 1.5 but subsequently the Food and Drug Administration (FDA) has approved a lower ODI cut-off of 0.5 [24,25]. Bio-Rad has very recently released a new assay which has been validated for testing on BAL as well as serum. There are no published data comparing both GM assays, unlike the comparison of the various Platelia Candida mannan assays.
The GM-EIA has been extensively examined for the earlier and more accurate diagnosis of IA in hematology patients. The sensitivity has varied between studies from 33 % to 100 % [24]. Like BDG analysis, this variation is related to differences in study design. In a meta-analysis the overall sensitivity was determined as 71 % with a specificity of 89 % [26]. The PPV is low (26-53 %) but the assay has an excellent NPV (95-98 %), indicating it may have more value as a screening tool to exclude IA. Some studies have also reported that GM can be detected a median of 5-8 days before culture positivity or clinical or radiological signs are present [27]. As a result it has been included as a microbiological criterion in the revised EORTC/MSG definitions of IA [13].
Whilst an ODI cut-off for positivity of 0.5 for serum is generally recommended it is less clear as to how many positive serum samples are needed to classify a case as IA. Maertens et al., found using ROC analysis that a cut-off of 0.5 in two sequential samples was associated with an optimal sensitivity of 92.1 % [25]. As a result, it is recommended that a positive result (ODI>0.5 in serum) indicating IA should be verified by retesting another aliquot of the same sample or obtaining a new sample and demonstrating reproducibility. Furthermore, studies have also indicated that the Platelia Aspergillus EIA may be useful for determining fungal burden and responses to antifungal therapy [28,29]. The ODI values over time are strongly correlated with treatment outcomes. Miceli et al., reported that the κ correlation coefficient for the ODI and survival was significant at 0.87 (95 % confidence interval 0.814-0.93; p<0.001) [29].
The sensitivity of the GM-EIA is lower in solid organ transplant recipients as compared with neutropenic and HSCT recipients. The assay had a reported sensitivity of 56 % in liver transplant recipients and only 30 % in lung transplant recipients indicating that this assay when used in serum may not have a diagnostic role in these populations [30,31]. The assay has also been examined in serum samples from HIV infected patients diagnosed with penicilliosis (15), cryptococcosis (22) and 11 HIV infected patients with no IFI (controls) [32]. The ODI was significantly elevated in those with penicilliosis indicating that the test may have a role for the early diagnosis of this infection in endemic areas.
The GM-EIA has also been evaluated in BAL. A metaanalysis of 13 studies demonstrated that at a cut-off ODI of 0.5 the pooled diagnostic odds ratio (DOR), sensitivity, specificity, positive and negative LR for proven or probable IA of 52.7, 87 %, 89 %, 8.0, 0.15, respectively [33••]. When a cut-off ODI of 1.0 was used the DOR (112.7), specificity (95 %) and positive LR (17.0) increased with similar sensitivity (86 %) and negative LR (0.15), indicating that for BAL the optimal cut-off is 1.0. In addition, the sensitivity of GM-EIA was higher in BAL as compared with its use in serum (65 %) [33••].
Mold-active antifungal agents may decrease the performance of GM-EIA causing false negative results [34]. False positive results may occur in those on antibiotics (especially piperacillin-tazobactam or amoxicillin-clavulanate), dialysis, Plasmalyte solution, other fungal infections, myeloma and neonates.
Cryptococcal Antigen-Based Assays
Cryptococcal polysaccharide antigens can be detected in serum and CSF using latex agglutination (LA) or EIA methods which are more sensitive than microscopy and culture (93 % for the LA compared with 50-80 % for microscopy) [35][36][37][38]. The LA required minimal laboratory infrastructure and is easy to perform. Its distinct disadvantage is its subjectivity related to reading the agglutination reaction. The EIA is as sensitive as the LA. The EIA has advantages over the LA. The EIA does not need pretreatment of serum samples and has greater reproducibility as results are generated objectively using a spectrophotometer [35][36][37]. However, EIA requires technical expertise to perform and more advanced laboratory infrastructure. It is best suited to larger reference and diagnostic laboratories.
More recently, a new cryptococcal antigen assay has been developed, namely, the lateral flow assay (LFA; Immuno-Mycologics, Norman, OK, USA). The LFA detects cryptococcal antigen by dipping an immunochromatographic test strip into a drop of diluted serum or CSF and incubating for 10 minutes. A positive result is when two bands develop in the test zone. The LFA has comparable sensitivity and specificity to the LA and EIA and has received approval from regulatory authorities both in Europe and USA for use in serum [39••]. The LFA may play a substantial role in the management of CM in developing countries. It requires minimal training, can be performed by non-laboratory staff and the assay requires no refrigeration. In addition, Immuno-Mycologics have adopted a global access pricing strategy such that it will be affordable in developing countries.
Molecular-Based Diagnosis of IFI
The PCR assays have the potential for the rapid, sensitive and early diagnosis of IFI. Many Candida-and Aspergillusspecific as well as panfungal assays have been developed for use in blood and other clinical specimens. Detailed discussions of different PCR methods can be found in in another recent review [40].
A meta-analysis of the use of PCR to diagnose IC has reported a sensitivity ranging from 73-100 % and a specificity of 92-100 %, with pooled sensitivity and specificity exceeding 90 % [41]. In this meta-analysis it was noted that higher sensitivities were achieved by using whole blood rather than serum samples, a commercial DNA extraction kit, panfungal ribosomal RNA or multi-copy gene targets and Candida-or fungal-specific PCR rather than multiplex assays. Specificity was increased by testing serial samples. Serial sampling and using consecutive results may assist in differentiating between colonization and true invasive infection. Similar to other non-culture-based methods it appears that PCR for IC diagnosis can make the diagnosis earlier than blood cultures (median of three days) [42].
Aspergillus PCR assays have been used for screening high-risk hematology patients with varying sensitivities (63-100%). A meta-analysis of Aspergillus PCR assays for use in blood has reported that high-risk hematology patients should be screened twice-weekly and that a single PCRnegative result rules out IA but to confirm IA two consecutively positive PCR results are recommended [43]. The variation in sensitivity is not only related to study design but also to the design of the PCR assay. A number of technical factors have been determined as affecting sensitivity and include DNA extraction methods (e.g., use of bead-beating), and volume of blood tested [44, 45•, 46]. The diagnosis of IA by Aspergillus PCR assays has preceded the diagnosis of IA by other methods (e.g., high resolution computed tomography (HRCT) scan and culture/ histological diagnosis by up to 3 weeks) [13,47,48].
Aspergillus PCR assays have also been examined and compared to GM in BAL specimens [49••]. The ROC indicated that a GM-ODI of 0.5 was optimal. Using this ODI, sensitivity and specificity was higher for GM-EIA than for Aspergillus PCR (79 % vs. 59 % and 96 % vs. 87 %, respectively). Use of positive results from both assays to define IA was associated with a sensitivity of 55 % and a specificity of 100 %. If either assay could be used to define IA, then the sensitivity and specificity were calculated at 83 % and 83 %, respectively. This indicates that if both tests are positive, then IA is effectively confirmed and that the combined use of both tests may improve the diagnosis of IFI.
There are conflicting data in the literature as to the effect of antifungal therapy on the sensitivity of PCR assays in both blood and BAL samples. Most report that Aspergillusactive antifungal therapy reduces the sensitivity but others have reported no effect [50][51][52][53][54][55][56]. This may be due, in part, to the form that Aspergillus DNA circulates in the blood and to the DNA extraction methodology. The PCR assays are particularly prone to contamination by air-borne spores which may result in false-positivity. Thus, a number of procedures need to be implemented in the laboratory to minimize contamination and the results need to be interpreted in the context of clinical and radiological findings.
A commercial Aspergillus PCR assay is available [57]. In comparison to a validated in-house assay it has a sensitivity of 60-70 % and a specificity of 90.5-100 %. The current limitations of fungal PCR assays are that they are not standardized nor have they undergone extensive multi-center clinical evaluation. As a result they are currently not included as a mycological criterion in the EORTC/MSG definitions of IFI. However, the European Aspergillus PCR Initiative has made significant advances in the area of standardization providing recommendations for extraction of Aspergillus DNA from whole blood [46,47].
Early Treatment Strategies for IFI
A randomized controlled trial (RCT) comparing a DDstrategy that guided the administration of anidulafungin therapy based on a positive BDG result (>80 pg/ml; Fungitell assay) to the administration of anidulafungin empirically based on physician's preference reported that BDG levels were significantly higher in those with an IFI versus those who had no IFI (117 vs. 28 pg/ml; p<0.001) [58]. Analysis of test performance indicated that the use of two sequential BDG results >80 pg/ml had a sensitivity, specificity, PPV and NPV of 100 %, 75 %, 30 % and 100 %, respectively. Twenty-one DD-strategy arm patients received anidulafungin as compared to five empiric arm patients. Anidulafungin was well tolerated and safe. However, a comparison of mortality outcomes was not reported for this trial. Two additional RCT have been completed but the results are not published to date (MSG-01 and INTENSE, www.clinicaltrials.gov). These trials may provide data to help determine the best management strategies for IC in the ICU.
Several DD-strategies for IA have been examined. A number of prospective non-comparative studies have been performed to determine the feasibility of a more targeted antifungal approach based on positive non-culture based assays or characteristic imaging findings rather than the traditional empiric approach of administering antifungal therapy to all with persistent or recurrent fevers despite broad-spectrum antibiotics [59][60][61][62]. These studies indicate that DD-driven strategies can reduce empiric antifungal therapy (EAFT) by up to 78 % (relative reduction), have an excellent NPV (100 %), don't miss cases of IA, can reduce antifungal drug costs (by £52, 839) and identify cases of IFI even in the absence of persistent fevers. However, these data only provide an indirect estimate of the impact of DD-strategies.
A number of RCT have been performed comparing a DDstrategy to the traditional EAFT approach. Cordonnier et al., enrolled 293 patients including low-risk patients undergoing autologous HSCT [63]. Significantly more IFI were diagnosed in the DD-strategy arm (13 vs. 4; p<0.02) but overall survival was no different between the arms (95 % vs. 97 %; p=0.12). On sub-group analysis, in those receiving induction chemotherapy, non-inferiority in terms of overall survival was not demonstrated in the DD-strategy arm. Overall, there was a significant reduction in antifungal drug costs in the DDstrategy (€1470 vs. €2252; p<0.001). In addition, a significant delay was detected between fever onset and initiation of antifungal therapy in the DD-strategy arm (median 13 vs. 7 days; p=0.01). However, this may be related to the delay in instituting the DD-strategy until 96-hours of febrile neutropenia despite broad-spectrum antibiotics. Hebart et al., enrolled 403 allogeneic HSCT recipients and randomized them to a PCRbased DD-strategy versus the traditional EAFT approach [64]. In the PCR-based arm, antifungal therapy was administered if one PCR assay was positive or if PCR negative they had persistent febrile neutropenia despite broad-spectrum antibiotics for five days. Significantly more antifungal therapy was administered in the DD-strategy arm (112 vs. 76; p<0.001) due, in the main, to the lack of specificity of the DD-strategy used in this trial. No difference in the number of cases of proven and probable IFI was detected between the arms. Whilst a significant mortality benefit was seen for the DD-strategy at day 30 post allogeneic HSCT, this was not evident at day 100 post transplantation. This finding may be explained by the lack of intensive monitoring (twice weekly) with PCR beyond day 30. Two further RCT have been published; one reported that a GM-based DD-strategy reduced EAFT use without decreasing survival and the second reported that PCR was a poor indictor of IA early after non-myeloablative SCT. However, both were small and inadequately powered [65,66].
Overall, a DD-strategy will replace the EAFT approach and provides a suitable alternative to broad-spectrum mould-active prophylaxis but questions regarding types of non-culture-based assays that should be used, the patient groups that a DD-strategy should be used in, timing of implementation of, duration of screening with a DDstrategy and cost-effectiveness still need to be answered.
Early diagnosis of CM allowing for early treatment appears to be the most practical approach in resourcelimited settings. Such a strategy involves screening HIVinfected patients with CD4 counts <100 cells/μL using one of the cryptococcal antigen assays (i.e., LA, EIA or LFA). If one of these assays is positive in an asymptomatic patient, then therapy using fluconazole should be instituted. Such a strategy has been studied in Uganda and has been shown to be associated a survival benefit (71 % at 30 months vs. 0 % if untreated) [67•]. Further, it has been estimated that such a screening strategy would be cost-effective in the setting of a prevalence of asymptomatic cryptococcal antigenemia of greater than 3 %. Currently, the prevalence of cryptococcal antigenemia in sub-Saharan Africa and South-East Asia is between 6 and 13 % [68,69]. Micol et al., compared primary prophylaxis to a screening approach and found that primary prophylaxis was more effective when the CD4 count was less than 50 cells/ μL, but screening was a more cost-effective strategy when the CD4 count was less than 100 cells/μL [69]. The availability of the new LFA, especially when used as a point-of-care test, may increase the scope of such a screening strategy by increasing the availability of the assay and decreasing losses-to-follow-up. However, before the widespread roll-out of such a screening strategy occurs a number of barriers need to be overcome including the training of health-care workers in the use of the LFA and the administration of fluconazole.
Conclusions
Numerous non-culture-based assays are now widely available for the diagnosis of IFI. Now, the challenge is how we integrate these assays into algorithms so that we can best target antifungal therapy to those who have an IFI and prevent unnecessary antifungal therapy in those without an IFI on one hand and on the other hand reduce mortality through earlier diagnosis.
Disclosure statement C.O. Morrissey has been a member of advisory boards for, received investigator-initiated grants from, and given lectures for Gilead Sciences, Pfizer, Merck, Sharp and Dohme and Orphan Australia.
Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. | 2018-04-03T02:09:17.543Z | 2013-01-11T00:00:00.000 | {
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14167406 | pes2o/s2orc | v3-fos-license | A scenario for leptogenesis at the TeV scale
We propose a scenario of thermal leptogenesis at the TeV scale in the context of an extension of the Standard Model with 4 generations. We also add one right-handed Majorana neutrino for each generation to generate left handed neutrino masses via see-saw. The r\^{o}le of the fourth lepton doublet is not much different from that of a supersymmetric partner, so the leptogenesis consequences of a four generation scenario are indicators of a more generic picture. We obtain a value for the baryon asymmetry of the Universe in accordance with observations by solving the full set of coupled Boltzmann equations for this model.
We have so far two experimental evidences to call for physics beyond the standard model (SM): the neutrino oscillations and the baryonic asymmetry of the universe (BAU).The SM with righthanded (RH) neutrinos (ν) provides an elegant mechanism for thermal leptogenesis.There are two intertwined requirements: reproduce the spectrum for light ν in the observed range and generate enough CP asymmetry through the out-of-equilibrium decay of heavy RH ν ( [3]).This asymmetry can be transmitted to the baryonic sector through sphaleron induced processes to explain the BAU.To achieve these two requirements, the RH ν should have Majorana masses and they should couple to the left-handed (LH) lepton doublets and the SM Higgs via complex Yukawa.The scenario of leptogenesis occurring at the TeV scale has been investigated for example in references ( [1,2], [4]- [6]).This scale is accessible in ongoing and near future colliders.Moreover, interesting new physics (like supersymmetry, extra dimensions, etc) could be revealed around that scale.It is now known that with 3 RH neutrinos it is difficult to achieve TeV scale leptogenesis and reproduce at the same time the small LH ν masses [1], unless one considers quasi-degenerate Majorana ν [4] or small fine-tuning [5].There are two approaches in the literature to realise TeV scale leptogenesis: (a) consider a quasi-degenerate spectrum of heavy RH ν and enhance CP asymmetry through resonant effects [7]; (b) extend the phase space parameters, either (i) by admiting, for example, extra couplings that allow three body decays of the RH ν leading to an enhancement of CP asymmetry [8], or, (ii) by extending the particle content.As regards the latter possibility, one may adopt among others either of two approaches: (1) consider a supersymmetric framework [9], (2) minimally extend the SM by having a fourth chiral generation and add a heavy RH ν for each of the four generations, assuming that the lepton asymmetry is due to the decay of the lightest RH ν (N 1 ) in the TeV scale [1,2].The scenario we present here is precisely this last approach.It is a simple extension of the Fukugita-Yanagida model [3] including one more generation to the SM.We will show how this simple model which can explain the BAU and provide appropriate values for ν masses and mixing angles.It is based on the out-of-equilibrium decay of a RH ν, in a C and CP violating way, such that a leptonic asymmetry is produced.The latter is converted into a BAU due to (B + L)-violating sphaleron interactions which are in equilibrium above the electroweak breaking scale.
The scenario and its constraints
The relevant part of the Lagrangian of the model we consider is given by, where ψ R i are 2-component spinors describing the RH neutrinos and we define a Majorana 4- component spinor, Our index i runs from 1 to 4, and α = e, µ, τ, σ .The σ component of L α corresponds to a LH lepton doublet which must satisfy the LEP constraints from the Z-width on a fourth LH ν [10].The λ ν iα are Yukawa couplings and the field φ is the SM Higgs boson doublet with vev v.We work in the basis in which the mass matrix for the RH ν M is diagonal and real, M = diag(M 1 , M 2 , M 3 , M 4 ) and define m D = λ ν v.To leading order the induced see-saw ν mass matrix for the LH ν is given by, m ν = λ ν † M −1 λ ν v 2 which is diagonalized by the well known PMNS matrix.
We consider the out-of-equilibrium decay of the lightest of the gauge singlets N 1 .There are two key points: (a) the CP asymmetry PoS(HEP2005)171 where f is a loop factor, has to be magnified by the presence of a large Yukawa coupling, and (b) the condition of out-of-equilibrium decay of N 1 has to be ensured, i.e.,Γ , where H is the Hubble expansion rate.These conditions restrict the size of the Yukawa couplings.
Another feature of our scenario is that the Yukawa coupling of the heavy LH neutrino is unsupressed (order 1) to ensure that the masses of the fourth generation leptons are heavy enough, and also to give an enhancement to the value of the CP-asymmetry (1.2).However, we note that the ∆L = 2 processes involving the fourth family of leptons in external legs are rapid and hence in thermal equilibrium.Consequently, the lepton asymmetry in the fourth leptonic direction is washed out.This changes the chemical equilibrium equations such that the relationship between B and L is modified to be: where Y L is the produced leptonic asymmetry only for the light active flavours.
Boltzmann Equations and Results
The evolution of the abundance of the N 1 , Y N 1 , and the lepton asymmetry Y L are given by ) where z = M 1 T , s is the entropy density and γ D j , γ S j are the interaction rates for the decay and ∆L = 1 scattering contributions, respectively.γ W is a function of γ D j and γ S j and ∆L = 2 interaction rate processes, called the washout factor which is responsible for the damping of the produced asymmetry.In eqs.(2.1) and (2.2), Y eq i is the equilibrium number density of a particle i. Explicit expressions of the interaction rates γ D j , γ S j and γ W are given in details in [2].An important comment is in order.The net leptonic asymmetry is produced only in the first 3 flavours as the strong ∆L = 2 process involving the fourth generation washes out the asymmetry in this direction.
In figure 1, we illustrate for a given set of input parameters the different thermally averaged reaction rates contributing to BE as a function of z = M 1 T : It is clear from this plot that for this set of parameters, and this is true for a wide range of parameter space, all rates at z = 1 fulfill the out-of-equilibrium condition (i.e.Γ X < H(z = 1)), and so the expected washout effect due to the ∆L = 2 processes will be small.The generated value of the BAU is η B ≃ 6 × 10 −10 .Applying the see-saw mechanism to our model for the chosen values of the parameters, we obtain m ν 4 > 48 GeV, and the three light ν masses are of the order of 10 −1 eV to a few 10 −7 eV.
Conclusions
This scenario is an extension of the SM by invoking a fourth chiral family, the leptogenesis effect of which is a generic indicator of many a physics beyond the SM, e.g., supersymmetry.We have presented the solutions to the coupled system of BE for our TeV scale model of thermal leptogenesis.We have carefully considered the effect of the interactions involving the heavy fourth generation leptonic fields and consistently written the BE which contribute to the final BAU together with the appropriate conversion factor from the lepton asymmetry to the baryonic one.Our results show that in this simple extension of the SM it is possible to produce the right amount of the BAU in a generic way.
Figure 1 :
Figure 1: Thermally averaged decay rates normalized to the expansion rate of the Universe H(z = 1) (left).Abundance Y N 1 and the baryon asymmetry Y B−L (right). | 2014-10-01T00:00:00.000Z | 2005-12-01T00:00:00.000 | {
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92988565 | pes2o/s2orc | v3-fos-license | Super Antibiotics , Part IV . Hyperforin , Relative and Absolute Stereochemistry Elucidated by Gas-Chromatography Mass-Spectrometry with Supersonic Molecular Beams
Relative and absolute stereochemistry for hyperforin has been proposed by Brondz et al. was supported and new isomer of hyperforin-perforatrin has been discovered. Relative and absolute stereochemistry for perforatrin was proposed and elucidated by using gas-chromatography mass-spectrometry with supersonic molecular beams (SMB). The possibility of the existence of ambiguities in chirality as a hypothesis for the existence, and a new understanding, of chiral substances with shifting chirality is presented.
Introduction
Hyperforin is one of the pharmaceutical active substances (PASs) in the plant Hypericum perforatum L., family Hypericaceae (Guttiferae).Hyperforin is a biologically active secondary metabolite in a class of prenylated phloroglucinol derivatives.Besides hyperforin, there are many other substances in genus Hypericum that are PASs which have the basic structure of hyperforin.The first use of hyperforin was as an antibacterial substance from extracts of H. perforatum L.
Hyperforin is now also medically accepted as an antidepressant.Hyperforin has multiple biological activities as medically useful PAS.Useful biological effects of hyperforin have been reported as being antibiotic, antifungal, antiviral, antiprotozoal, anticancer, immunomodulating, and others.Genera such as Garcinia, Clusia, Symphonia, Allanblackia, and Calophyllum also contain polyprenylated acylphloroglucinols, the main block of which contains hyperforin, as a structural basis.All these substances possess useful biological activities.Knowledge about the exact relative and absolute stereochemistry of hyperforin is important.Some confusion now exists regarding the relative and absolute structure of hyperforin introduced by Bystrov The necessity for tools in the struggle against infectious diseases became obvious at the beginning of the twentieth century.Massive epidemics that followed World War I heavily supported this idea.In the years 1920-1925, several publications describing observations in this field were presented [1] [2].In 1928, the Scottish microbiologist Alexander Fleming observed antagonism of a mold toward Staphylococcus colonies in a petri dish [3].In the former U.S.S.R., attention was directed towards antibacterial substances from plants [4].Already during World War II, extracts from H. perforatum were used there.One of the antibacterial substances in the extract of H. perforatum was described as hyperforin [5].
Significant efforts were undertaken by the research team of Bystrov et al.
In [6], they stated that hyperforin has a configuration of 6S and 7R, but in [10] and [11], they stated that it has a configuration of 7R and 8S-they changed their mind.The chiral centers C 1 and C 5 [6] were also relocated to C 5 and C 1, respectively, in [10] and [11].In these three papers ( [6] [10], and [11]), the chi- presented in [6] [10], and [11] are now shown here in Figure 1 (structures reproduced from the three references mentioned).The numbers in red have been Int.J. Analytical Mass Spectrometry and Chromatography and [11].(Numbering of carbon atoms in red and naming of structures following IUPAC rules were done by the author of this paper).
Up [12].The antibacterial spectrum and mass spectrum of hyperforin were also studied, and reported in [12].The composition of H. perforatum was reviewed in [13] and [14].The first mass spectrum of hyperforin, recorded using chemical ionization, was obtained in 1978 and published in 1979 [12] (see Figure 2).Using X-ray crystallography, the relative and absolute stereochemistry of hyperforin has been elucidated, as reported in [15] and [16], respectively.Figure 3 shows the structure of the p-bromobenzoate ester of hyperforin (1) and hyperforin (2).Hyperforin is a thermolabile substance that is easily oxidized by oxygen or air.
At the time of X-ray analysis of the relative configuration [15] and absolute configuration [16], it was not yet being possible to undertake chromatography-mass spectrometry analysis to confirm the purity of crystals.The oxidative analysis of crystals gave 95% and 97% compliance with dinitro-benzoate of hyperforin [15] and p-bromobenzoate of hyperforin [16], respectively.Quantities of impurities present in crystals did not interfere with the results of X-ray analysis.
A sample of the p-bromobenzoate ester of hyperforin was placed in a glass ampule under Ar gas (high quality; Norsk Hydro AS, Norway), the ampule was soldered and then capped in liquid nitrogen until required for the gas-chromatography mass spectral analysis described in the present paper.Results of GC-MS with SMB analyses are shown in figures presented in the next section (Materials and Methods).
Materials and Methods
The above sample of transparent white crystals of the p-bromobenzoate ester of hyperforin (described in [16]) (1.0 mg) was dissolved in n-hexane (1 mL; analytical grade reagent, Merck, Darmstadt, Germany).GC-MS analysis with supersonic molecular beams (SMB) instrumentation enabling the separation and analysis of thermolabile substances is described in detail in [17] and [18].The actual instrumentation used here for GC-MS with SMB was based on a Varian 1200 GC-MS system (Varian, Middleburg, The Netherlands); details are described in [19].Separation of substances and resolution of isomers was carried , and [11].
Results and Discussion
Hyperforin in free form is a keto-enol molecule, which is subjected to keto- Rapid tautomeric equilibrium between a keto form (a ketone or an aldehyde) and an enol (an alcohol) (see Figure 8), which contains a pair of doubly bonded carbon atoms adjacent to a hydroxyl (−OH) group, is highly thermodynamically driven (see Figure 9) and specific pH can favor one of the tautomeric forms.
During the application of a molecule of hyperforin at room temperature and in the presence of derivatizing agents, the conditions are favorable for the derivative (1) in Figure 3 as a major product.However, a minor derivative A was also present in a TIC chromatogram (Figure 4) and a mass spectrum (Figure 5).Use of a different pH from that used in derivatization for the described p-bromobenzoate ester of hyperforin major isomer B could lead to the minor isomer A.
The production of derivative A could be explained, as shown in Figure 10 and
Stereochemistry of Hyperforin Proposed by Brondz
X-ray crystallographic analysis is generally accepted as proof of the stereochemistry of previously unknown molecules.However, in order to finalize any discussions, it is important to obtain independent evidence and decisive proof.The In 2013, Shair et al. reported on the results of the enantioselective synthesis of hyperforin in J. Am.Chem.Soc.[20].The structure is presented there, and reproduced here in Figure 12, with the addition of the authors' numbering of the C atoms, following IUPAC conventions for the numbering of C atoms, and their chirality.
In 2010, a paper entitled 'Catalytic Asymmetric Total Synthesis of ent-Hyperforin' by Yohei Shimizu et al. was published [21].In it, the authors state the following: " 1 H, 13 C NMR, and IR spectroscopic data as well as mass spectrometric data were all identical with the reported values.The optical rotation of synthesized 5 was opposite to that of the natural isomer ([a] D 23 = -36.8(c = 0.38, hyperforin in [16], and (4) hyperforin in [16] in two-dimensional projection.
to present new, however incorrect (because of IUPAC rules), renumbering of C atoms for the previous structure published in [6] by new numbering publishing in [10] and [11], it is obvious that this is the same structure as presented in 1975 I. Brondz [6]; the structure is also incorrect in terms of both relative and absolute stereochemistry.Further explanations are given in [22].
General Discussion
It is important to compare the structure of hyperforin published by Bystrov et al. [6], which is identical to the structure given in [11], with the major B [15], [16] (see Figure 14).and minor A isomers of hyperforin to demonstrate differences (see Figure 15).Here it is obvious that there [6] and [16] are two different molecules, if the numbering of the C atoms in both molecules follows IUPAC rules.
To demonstrate this more clearly, both molecules were divided by arrows on the left (L) and right (R) sides.In molecule (1), the C-6 position is not substituted and is on the left side, while in molecule (2), the C-6 position is substituted and on the right side.In both molecules, the isobutyryl, methyl, and methylpenthyl substituents are from the right side; in molecule (1) the hydroxyl is from the left, but in molecule (2), the hydroxyl is from the right.The keto substituent in molecule ( 1) is from the right, but in molecule (2), the keto substituent is from the left.Double bonds in the molecules are on opposite sides to each other.From all these described differences, it is clear that we have two different structures, even in terms of relative stereochemistry.This clearly indicates that the structures of Bystrov [6] [11] have nothing in common with the major B isomer of hyperforin [16].
A comparison of the hyperforin' structure of Bystrov with that of the minor A isomer of hyperforin-perforatrin is shown in Figure 15.could not repair the structure presented in 1975 [6] because the major B isomer of hyperforin as it was shown in [15] and [16], and in Figure 14 is different from structure presented in [6] [10] and [11] in relative as well as in absolute stereochemistry, the perforatrin is diasteriomeric to proposed by Bystrov et al., structure of hyperforin in relation to chirality of C 1 and C 5 and stated in [11] 7R and 8S configuration.
In publication [23] was adopted the formula of major B isomer of hyperforin as it presented in Figure 3, and formula of hyperforin Figure 12 as was presented in [20], and formula of hyperforin Figure 13 as was presented in [21].In all of these publications, the same relative and absolute configuration for the major isomer of hyperforin is 1R, 5R, 7S, 8R, which follows from publications [15] and [16].Figure 16 also shows the structure of hyperforin from the publications of Bystrov et al. Figure 16(2).A more conventional IUPAC interpretation Figure 16(2) is presented as Figure 16(3).Structure (4) in Figure 16 is the result of an attempt to redraw structure Figure 16(3) according to the style used in The Merck Index and in Wikipedia.
The first time that the structure of hyperforin was presented in The Merck Index [24] and later in [25] and in Wikipedia, it was reproduced as structure (5) in Figure 16, it is different in chirality from Figure 16(4).
In Figure 16, (6) presents the transformation of Figure 16(1) by the fashion 5) is the structure presented in The Merck Index [24] [25] and in Wikipedia, and ( 6) is the transformation of (1) by the fashion used in The Merck Index (2001) [24] and in Wikipedia.
In 2002, Petra Adam et al. published [26] (see Figure 17) in which the structure of hyperforin was elucidated from biosynthesis of hyperforin in H. perforatum "Hyperforin was isolated and analyzed by quantitative NMR spectroscopy" [26].
Graphically presented structure was not furnished with a description of its stereochemistry, however, from the graphical representation and by applying IUPAC rules, it was possible to arrive at the following stereochemical name:
Conclusions
Hyperforin has four chiral centers and, theoretically, the existence of 16 different stereoisomers is possible.However, because hyperforin is a secondary metabolite of plant origin and in its biosynthesis the enzymatic reactions take place, the product is highly enantiospecific.For this reason, only one isomer of hyperforin should be expected.However, there are two stable isomers that exist in a dynamic equilibrium because the molecule is a tautomer.Evidence of this was provided by GC-MS with SMB analysis.There are similar cases expected and will be discovered in tautomeric molecules with multiple chiral centers.
It has been proven that the structures of hyperforin as described by Bystrov et al. differ from the major B [16] isomer of hyperforin and from the minor A iso- mer of hyperforin-perforatrin in terms of both relative and absolute stereochemistry.
The stereochemical structure of hyperforin published in [15] [16] by Brondz et al. is supported by [20] [21] and [26], and it is in compliance with major B isomer of hyperforin presented in this paper.The major pool of hyperforin in plant H. perforatum exists as isomer B [26] earlier elucidated by X-ray crystallography in [15] and [16].Perforatrin minor A isomer was discovered by GC-MS with SMB and was described in this paper.
et al.Mistakes of Bystrov et al. are discussed in a paper "Super Antibiotics, Part III.Hyperforin, Revision of the Relative and Absolute Stereochemistry Presented by Bystrov et al.".Further elaborations of the relative and absolute stereochemistry of hyperforin are necessary.The additional gas chromatography-mass spectrometry with supersonic molecular beams (GC-MS with SMB) evidence recorded by Brondz, and presented here, should clarify the subject.Possible reasons for the mistakes made by Bystrov et al. concerning the relative and absolute stereochemistry of hyperforin are discussed, and GC-MS with SMB evidence for the existence of isomer to hyperforin-perforatrin-is presented.Perforatrin is a new diastereomer (isomer) of hyperforin.The possibility of the existence of ambiguities in chirality as a hypothesis for the existence, and a new understanding, of chiral substances with shifting chirality is presented in the paper.
figures presented in these publications and for more information (see in a paper 'Super Antibiotics: Part III.Hyperforin, Revision of the Relative and Absolute Stereochemistry Presented by Bystrov et al.').Figures of the hyperforin structure
out on a VF-5HT column (Varian, Middleburg, The Netherlands): 4 m length, 0.25 mm i. d., 0.1 µm film thickness.The reduction of column length was done in the laboratory.The flow rate of helium in the column was 8 mL/min.A sample extract in n-hexane (1 µL; approximate concentration 100 ppm) was injected (split ratio of 10:1), using the Varian 1079 injector, at 230˚C.The GC oven was programmed from 120˚C to 300˚C at 20˚C/min.Separation of substances, the resolution of isomers, and the detection of compounds in crystals of the p-bromobenzoate ester of hyperforin are shown as a TIC chromatogram in Figure 4.The MS fragmentation of substances of MS peaks is shown in Figure 5 and Figure 6.
Figure 7
Figure7shows the structure of the p-bromobenzoate ester of hyperforin[16], together with the percentage composition of elements in crystals analyzed by oxidative analysis and the theoretical calculated Molecular Weight (MW) (in Dalton) and mass m/z of M + .Bromine 35 Br has 30 known radioisotopes, however, only two stable isotopes are known: 79 Br and 81 Br.For this reason, the masses m/z 719, 720, and 721 are present in the mass spectrum of the p-bromobenzoate ester of hyperforin.The appearance of two isomers, A and B, of hyperforin supports the correctness of the relative and absolute stereochemistry of the molecule[15] [16], and can partially explain the mistakes in the publications of Bystrov et al.; in reports[6] [10], and[11].
Figure 4 .
Figure 4. Composition of p-bromobenzoate ester of hyperforin crystals [16] shown as a TIC chromatogram.Peak A is the minor isomer of p-bromobenzoate ester of hyperforin, and peak B is the major isomer of p-bromobenzoate ester of hyperforin.
Figure 5 .
Figure 5. Mass spectrum of peak A, the minor isomer of the p-bromobenzoate ester of hyperforin.
Figure 6 .
Figure 6.Mass spectrum of peak B, the major isomer of the p-bromobenzoate ester of hyperforin.
Figure 7 .
Figure 7. p-Bromobenzoate ester of hyperforin [16] together with percentage composition of elements in crystals analyzed by oxidative analysis, and Molecular weight calculated (in Dalton) and mass m/z of M+.
Figure 9 .
Figure 9.A specific pH can favor one of the tautomeric forms.
Figure 11 .
Figure 11. Figure 11 shows the production of the p-bromobenzoate ester of hyperforin as the minor derivative A. The separation of these two stereomers, A and B, was possible with an achiral column because they are diastereomers and differ in terms of their relative structures.Diastereomers are substances that differ in terms of their physicochemical parameters; they have different melting and boiling points.The prevalence of the A or B isomer in natural hyperforin, and in the stomach, blood, lymph, and body treasures, can strongly depend on the pH and cations in electrolytes.Because isomer A of hyperforin is different from isomer B of hyperforin, I propose it be referred to as perforatrin.
Figure 10 .
Figure 10.All molecules shown could exist in the mixture together with underivatized major isomer hyperforin.
Figure 11 .
Figure 11.Production of p-bromobenzoate ester of hyperforin from minor derivative A.
Figure 12 .
Figure 12.Structure of hyperforin presented in [20].(Name of the substance and numbering of C atoms following IUPAC convention were added by the author of this paper).
Figure 16 .
Figure 16.Structures: (1) is the structure of hyperforin as published in a review by Paola Zanoli [23], (2) is the figure taken from the publications of Bystrov et al., (3) is the conventional IUPAC interpretation figure 17, (4) is an attempt to redraw (3) by the fashion used in The Merck Index (2001)[24] [25] and in Wikipedia, (5) is the structure presented in The Merck Index[24] [25] and in Wikipedia, and (6) is the transformation of (1) by the fashion used in The Merck Index (2001)[24] and in Wikipedia.
until 1978, Bystrov et al. published structures of hyperforin.Brondz et al.Brondz et al. notified Bystrov et al. that our data suggested errors in their claim regarding the stereochemistry of hyperforin.Results of Brondz et al. first reinvestigation into H. perforatum and hyperforin were presented in 1979 | 2019-03-31T23:24:44.779Z | 2017-09-13T00:00:00.000 | {
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267018430 | pes2o/s2orc | v3-fos-license | Living Life as a Text/tile: Animating Asian Americanist Reading Practices
ABSTRACT This is an experimental work based loosely around my stop-motion animation, “Scare Quotes.” It asks – what is it like to live life as a textile? By imagining the self as a textile in my visual practice and writing, I consider Ornamentalism as an embodied reading and creative practice. I perform constructions of Asian femininity to critique their role in the avant-garde poetry of Gertrude Stein and Susan Howe. By stitching together the work of Lisa Lowe, Anne Cheng, and Mel Chen in this performance, I envision Asian Americanist critique as poetry and as a mode of living and feeling.
I noticed that I was living my life as a textile.It happened while reading Susan Howe's The Midnight, her word squares materializing into a blanket.I climbed out of a well of words and used the blanket to dry myself off.A patchwork blanket, like me, made of texts and feelings.Howe threads bed hangings into word squares, curtains into quote fragments, photographed knots into stuck words.I think honest meanings live in fraying threads and the eyelets between stitches, in roughness, in abruptness.They whisper softly, I want to be unraveled I want you to unravel me I revel in your unraveling.I once built seams too.I moved scraps of paper millimeters at a time.I loved small feelings, not big ideas.I was not a thinking critic but a feeling textile.
I cut paper and cut paper and cut paper; my hands cramp up.It's dark in the animation studio.I place the pieces on a glass plane, I nudge my textiles softly, I click the camera.It's hard to refrain from big maneuvers.Like arguments, one broad gesture can obliterate what it aims to move.I'm not thinking straight, I'm thinking in exalted and halted undulations.I'm loosely fastening unwieldy emotions to small movements, wrapping ruptures in red and royal blue fabric.Hours of motion produce a seconds-long sequence of textiles swaying joltingly, as if they're afraid of moving yet dare to dance anyway.As if they're caught or cut in violence.It's resilient weakness.Sentenced to a thousand cuts, dyeing, not dead.Crimson and cobalt bleeding into each other joyously.Weightless, yet still moving with intention.
These memories resurface as I contemplated Howe's The Midnight.Even as I was repelled by Howe's suspension of silks and calicos and its dispersal of racial violence, I wanted to tease out "the impious history of sensation" and its enchanting "cobweb gossamer ephemera." 1 The curtains murmured in my ears, tethered to their billowy existence, and I wanted to speak back.I gathered Howe's texts in bundles and picked apart their threads to remake them.Their threads spun out into new associations.I thought of my recent readings and viewings -of intimate continents, of buttons that were tender, a bizarre market and its animacies.My research into the works of Susan Howe, Lisa Lowe, Gertrude Stein, Jodie Mack, Mel Chen dissolved into a single dream that I carried with me out of sleep and into life.As I read, words changed shape and color and began dancing before my eyes.I wanted to sustain these rough and transient squares and affix them to a screen.I wanted to be them -a beautiful thing flickering between living and pausing.Like light flares, the texts I read and films I watched shone on The Midnight.My reading of this single text was a bundling of past words, images, movements that stuck to me and became me.
I returned to stop-motion animation to flesh out this textilic existence -to understand why I see words as fabric patterns as myself, and how imagining myself as a textile indulges desire while skimming across its surface with a barely visible critical gloss.I move textiles and move with them so I can be them.Being a textile occasions a raveling and unraveling of the self through desire, a reorientation of the senses that pierces through racialized structures of feeling and their residual presence.Being a textile unfolds your body to be used, felt, shaped, and molded.Now unfurled, you loosen the hold of the personal and familial and communal and artistic histories you're bound in even as you risk re-entanglement.As a textile, you notice that desire lingers in the embrace of complicity.Being a textile means falling into your next move -an embrace, an escape, an unearthing of emergent forms of fabric dwelling.As I began to weave these associations into my philosophy of animation, I became more aware of the seams holding me together, thinking threads that bind me to my living reading of Howe.These four stitch-theories enchant me as I become a word square.In rows, they keep me composed and give shape to my overflowing form.
Stitch 1, draping, after Lisa Lowe
It begins with a dress form -a still, canvas body, bare as it (I) reveals and conceals.In The Intimacies of Four Continents, Lisa Lowe unwinds the gossamer spine of Orientalism upon which Victorian fantasies of romance rest.This spine runs through the window curtains, bed silks, and lavish organza fabrics in Thackeray's Vanity Fair.As Lowe describes, "The bed curtain, the 'chintz of a rich and fantastic Indian pattern, and doublé with calico of a tender rose colour,' both reveals and conceals" intertwined histories of colonial production. 2 This Orientalist spine also sutures Victorian literature to modernism.
In new time, Gertrude Stein reanimates the room.She reveals that everything is concealed, breaking apart language and its obscurity."Plates and a dinner set of colored china." 3 The enclosure of the feminized domestic space amplifies colonial murmurs: the ceramic "china" of Stein's button-filled domicile objectifies and reduces "china" to decorative commodity objects.The incomplete language of the nation transforms into the incomplete language of ceramic breakages.
There can be breakages in Japanese
No cup is broken in more places and mended, that is to say a plate is broken and mending does do that it shows that culture is Japanese 4 Stein's ceramic menagerie breaks apart one continent into its Orientalist literary fragments."China" is a euphemism for the shiny, decorative surface.The broken plate haphazardly and imprecisely evokes the Japanese process of kintsugi -mending broken pottery with golden lacquer -as a euphemism for the fragility of re-crafting something, perhaps with language.Josephine Park reads this reductive invocation of a regional craft as an imagined relic of "Old Japan" and its "rapidly disappearing world of charming antiquity." 5rientalist nostalgia is the beautiful face diverting attention from the imperial interests and anxieties of the text -economic investments in both China and Japan as well as fear for the white Western subject. 6These broken ceramics function as symbols of a fragmented and apparitional Asia.But amongst these shards, can I find a version of myself?I use textiles to transform these alienating reductions -the interchangeability Stein constructs between "china," Japan, ceramic object -into a more ambivalent permeability.
Passing between calicos and silks and linens, strobing film sequences animate the intimacies that Lowe maps out between the production of one continent and the limited desire of another.While holding and curating cute objects may exhilarate the feminized white speaker, the figure of the Yellow Woman 7 hardens into that held, cute object.She falls to pieces, held together, gold lines between her sensationalized shape.Yet I love this writing, I identify with these abject objects.As word slippages unfold through imperialist word play, I become the abject object to break apart myself to break apart this "china."Stein manufactures a quotidian lusciousness of Indian rugs, china plates, and other racialized commodities that echoes the realist imaginary of Thackeray's idyllic Victorian households.Tender Buttons has been read as a celebration of the taste for desire and difference. 8tein's intimate buffet of shattered grammar and sumptuous food includes and decenters racialized objects, lets them ripen and rot.As an animator, I move (with) origami paper and fabrics.My textiles resurrect the objects of The Midnight and Tender Buttons, their decorative energy escaping the supposed purposelessness of pretty.I lean closer, needling my desire to caress buttons and drapings despite their gossamer spine.With caution, Lowe embraces fabrics and observes as they "reveal and conceal" history. 9I touch and brush up against these fabrics.I enact historical quillwork through the sway of my silky body.Wrapping myself in gauzy archives and shattering alongside dazzling plates, I "reveal and conceal" history.My fabrics melt into words that repeat archaic patterns, sometimes mockingly and at times in yearning.
Stitch 2, transmogrification, after Jodie Mack
A stitch that attaches denim to velvet.A stitch that attaches text to textile.I observe the kinship between Howe's The Midnight and the fluttering visuals of the animators In Grand Bizarre, Mack's market of fabric, soft arrays form a strobe effect alternating between maps and textiles, alphabets and fabric patterns.This effect unfolds the difference between text and textile, like Howe's word squares. 11The textiles saunter and strobe, creating time colors.Embroidered flowers flicker from written signs to tattoos on the body to woven cotton to shimmering silk.These apparitions sing to each other, letting their light auras linger and overlap, and they cut each other off, obliterating their afterimage glow.I turn light into sound, a cotton frame into a silk frame, soft fabric into halted motion.
Stitch 3, surface spectacle, after Anne Cheng
Being a textile is being a material girl -a girl fashioned out of materials.It is feeling objectified and making something of it.Self-styling as animation.Anne Cheng elaborates a theory of Ornamentalism where excess decoration renders the flesh invisible and threatens to merge with it, shielding against violence while creating new, muted versions.For Cheng, Ornamentalism is neither complicit nor subversive.By letting textiles speak on my behalf, or rather, by shapeshifting into a speaking textile, I sink into my invisibility and the touch of terror-joy.A textile finds its own purpose in being used.I find joy in being used (sometimes I use myself, I myself am implicated).This stop-motion film animates and reformulates the themes and formal techniques that Howe uses to stage the dialogue between text and textile.Words appear as ghostly impressions upon colorful fabrics.Patterned squares weave in and out of Howe's word squares.I arrange cutouts as Howe "arranges snippets." 12erhaps the avant-garde recuperates cuteness and the feminine.But while inanimate objects become animate, their whispers remain stifled.Still, they trouble the hard surface of cute.Whispering objects encourage the reader to double back, search for synonyms, locate missing parts of speech.But what if I told you I was a tender button?And also a tender textile?I know what it feels like to be stared at, gaped at, moved, and severed.I identify with these texts -not with the speaker inhabiting a more-than-real domestic space with timid furniture and small recipes, but with the things in the narrative, the little pieces of texts as textiles and textiles in texts.Feeling held by these texts in cruel ways, I refuse to linger on the meaning of words or even the meaning of their sounds.Instead, I hear a swatch of fabric, its neatly woven colors intimate in their almost inscrutable audibility.I feel the fabric whisper upon my skin and I whisper back a refrain, our hushed voices melting into a soft, shiny surface.
Stitch 4, animating animacies, after Mel Chen
Animating animacies' proximity to objects, living as an object, being moved and moving, racial abjection.Mel Chen finds slippage between the human and the inhuman in their theory of animacy.They suggest that hierarchies of animacy maintain white male supremacy by placing minoritarian subjects in close proximity to animals, things, and concepts.The dominating structures that prescribe the meanings of personhood rely upon hierarchies between life and the non-living.As Chen writes, beings at the top of animate hierarchies are those most capable of affecting, while vegetality describes a "failure of lifelines, of ability to act upon others." 13But why should I, or you, avoid being acted upon?Chen argues that, while marginalized, the state of nonliving can be recuperated as a mode of being that transmutes the scary intimacies of intersubjectivity, of connecting to humanized objects and objectified humans.I seek to animate a textile of desire to reanimate myself.The joy of being moved.The terror of racial abjection.Simultaneously fluid and arresting, animation gives emotion a life beyond me.What can we enact through paper?Meaning is raveled and wound up again, stitched together and taken apart, a constitution of words and textiles rearranged simultaneously.Joy and terror puncture me, threading in and out of my inside self, through things, people, forming our attachments.
Using each of these four stitches, I animate myself as a collection of textiles and bring myself to life as a textile.Art practice merges abject objecthood as a state of being and desire as an act of doing.I channel the voice of a textile to contemplate my art practice, my scholarship, the scholarship that moves me and, simply, is me, and the desire that threads these mundane life practices together.I animate desire as a method of reading that leaves strands open, unhemmed, that invites you to spin the way you want, to allow myself -yourself-to become, instead, a living textile.This art practice and method of being a textile is a way of understanding the self as imbricated in deep and traumatic histories, the effects these histories have on stereotypes and archetypes, and most importantly, the urge to create and find ecstatic moments.Art enables an interplay between stasis and motion, being and doing, unliveliness and liveliness, labor and love.Being a textile is a mode of creating, understanding historical violence and the violence of the everyday, a way of living on without forgetting.Being a textile is being cut and torn apart, unraveled, but knowing how to sew oneself back together, perhaps roughly.Being a textile is to be open to attachment, detachment, movement, standing still only to come to life again.Being a textile is getting wrapped up in things until you must tear them apart until you must get knotty until you must separate seams.The difference is spreading.The difference is spreading.The difference is spreading. 14 | 2024-01-17T16:02:29.540Z | 2023-05-04T00:00:00.000 | {
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1322343 | pes2o/s2orc | v3-fos-license | E-2-hexenal promotes susceptibility to Pseudomonas syringae by activating jasmonic acid pathways in Arabidopsis
Green leaf volatiles (GLVs) are C6-molecules – alcohols, aldehydes, and esters – produced by plants upon herbivory or during pathogen infection. Exposure to this blend of volatiles induces defense-related responses in neighboring undamaged plants, thus assigning a role to GLVs in regulating plant defenses. Here we compared Arabidopsis thaliana ecotype Landsberg erecta (Ler) with a hydroperoxide lyase line, hpl1, unable to synthesize GLVs, for susceptibility to Pseudomonas syringae pv. tomato (DC3000). We found that the growth of DC3000 was significantly reduced in the hpl1 mutant. This phenomenon correlated with lower jasmonic acid (JA) levels and higher salicylic acid levels in the hpl1 mutant. Furthermore, upon infection, the JA-responsive genes VSP2 and LEC were only slightly or not induced, respectively, in hpl1. This suggests that the reduced growth of DC3000 in hpl1 plants is due to the constraint of JA-dependent responses. Treatment of hpl1 plants with E-2-hexenal, one of the more reactive GLVs, prior to infection with DC3000, resulted in increased growth of DC3000 in hpl1, thus complementing this mutant. Interestingly, the growth of DC3000 also increased in Ler plants treated with E-2-hexenal. This stronger growth was not dependent on the JA-signaling component MYC2, but on ORA59, an integrator of JA and ethylene signaling pathways, and on the production of coronatine by DC3000. GLVs may have multiple effects on plant–pathogen interactions, in this case reducing resistance to Pseudomonas syringae via JA and ORA59.
INTRODUCTION
Plants produce green leaf volatiles (GLVs), C6-aldehydes, C6alcohols, and their acetates, through the lipoxygenase (LOX) and hydroperoxide lyase (HPL) pathways. Linoleic and linolenic acid are the substrates for dioxygenation and subsequent cleavage to obtain C6-volatile aldehydes that can be further modified by alcohol dehydrogenases (ADH), an isomerization factor and an acetyltransferase leading to the formation of a bouquet of these volatiles. Intact plants produce only trace amounts of GLVs, whereas these compounds are rapidly emitted in large amounts after wounding, herbivory or pathogen attack (Croft et al., 1993;Turlings et al., 1995;Fall et al., 1999;Shiojiri et al., 2000Shiojiri et al., , 2006aHeiden et al., 2003).
www.frontiersin.org
Finally, GLVs possess fungicidal and bactericidal activity (Prost et al., 2005;Shiojiri et al., 2006b). Since GLVs are released after infection with pathogenic fungi and bacteria (Croft et al., 1993;Heiden et al., 2003;Shiojiri et al., 2006b), this suggests that a possible physiological role of these volatiles is to limit pathogen growth. Several observations support this hypothesis. For instance, upon infection with the pathogenic bacteria Pseudomonas syringae, Phaseolus vulgaris (lima bean) leaves release relatively high amounts of the C6-aldehyde E-2-hexenal and the C6-alcohol Z-3-hexenol (Croft et al., 1993). Moreover, pre-treatment with the C6-aldehyde E-2-hexenal as well as genetic manipulation to enhance C6-volatile production, resulted in increased resistance against the necrotrophic fungus Botrytis cinerea in Arabidopsis, most likely as a result of both activation of defense responses and direct inhibition of fungal growth (Kishimoto et al., 2005;Shiojiri et al., 2006b).
Since all this evidence indicates a role for GLVs in regulating plant responses to bacterial pathogens and GLV levels have been shown to increase in plants upon infection with Pseudomonas syringae (Croft et al., 1993;Heiden et al., 2003), we decided to further dissect the role of GLVs in the interaction of plants with this pathogen. Increased GLV levels could directly inhibit the pathogen and/or promote infection through downstream signaling favorable for the pathogen. Pseudomonas syringae pv. tomato DC3000 is a plant pathogen that enters leaves through stomata, multiplies in the apoplast, and produces necrotic lesions with chlorotic halos (Hirano and Upper, 2000). Pseudomonas syringae pv. tomato DC3000 (DC3000) causes bacterial speck on tomato (Cuppels, 1986), but also on A. thaliana (Whalen et al., 1991). DC3000 produces coronatine (COR), a toxin, responsible for chlorotic halos, which mimics the action of JA-isoleucine (JA-Ile), the active form of JA. With this phytotoxin DC3000 exploits the antagonistic interaction between JA and salicylic acid (SA) in order to shut down SA-dependent defenses that plant triggers to fight against Pseudomonas infections (Block et al., 2005;Glazebrook, 2005).
We especially focused on the role of E-2-hexenal during the Arabidopsis-Pseudomonas interaction. Although it is not the most abundant C6-volatile produced by HPL activity, E-2-hexenal is emitted during Pseudomonas ssp. infections in lima bean (Croft et al., 1993) and in tobacco (Heiden et al., 2003), and it has the highest bactericidal activity in vitro among oxylipins (Prost et al., 2005), likely because its α,β-unsaturated carbonyl moiety that can react with nucleophilic groups (Farmer and Davoine, 2007). Additionally, E-2-hexenal has been shown to induce several responses in Arabidopsis, including induction of defenses, inhibition of root growth and enhancement of resistance against the necrotrophic fungus B. cinerea (Bate and Rothstein, 1998;Kishimoto et al., 2005;Mirabella et al., 2008). In order to determine the role of GLVs in the responses against Pseudomonas, we set out to study Arabidopsis plants with and without a functional HPL (Shiojiri et al., 2012) and did complementation studies with E-2-hexenal. Remarkably we found that the presence of a working copy of HPL increased susceptibility of Arabidopsis to DC3000. Treatment with E-2-hexenal also enhanced the susceptibility to this bacterial pathogen. We found evidence that this is mediated by the transcription factor ORA59, one of the main players in the JA-signaling pathways, and required the production of the bacterial toxin COR.
BACTERIAL POPULATION COUNTS
Bacteria were grown overnight at 28 • C in liquid King's broth (KB) medium (King et al., 1954) containing rifampicin (50 μg/ml) for the Pseudomonas syringae pv. tomato DC3000 strain, and kanamycin (100 μg/ml) for the cor − DC3682 mutant strain, unable to produce COR (Ma et al., 1991). Plants were inoculated with either a low dose (OD 600 of 0.0007), for bacterial growth assays, or a high dose (OD 600 of 0.007), for qRT-PCR and hormone quantification, of the bacterial suspension, and bacteria (colony forming units, cfu) were counted as reported in Park et al. (2010).
PLANT HORMONES EXTRACTION AND QUANTIFICATION
For JA and SA quantification, 12 leaves were harvested, in pools of 4, from 12 different mock-infiltrated (10 mM MgSO 4 ) or bacteriainfiltrated plants in two independent experiments. To extract JA and SA, frozen leaf material (50-150 mg) was ground and homogenized in 0.5 ml 70% methanol, spiked with 200 ng of D6-JA and D6-SA (internal standards for extraction efficiency; CDN Isotopes, Canada 1 ), with a Precellys24 automated lyser (Bertin Technologies 2 ). Samples were homogenized twice by shaking at 6,000 rpm for 40 s and centrifuged at 10,000 g for 20 min at 4 • C. The supernatants of two extraction steps were pooled. Hormones were quantified by liquid chromatography-mass spectrometry (LC-MS) analysis on Varian 320 Triple Quad LC/MS/MS. Ten microliters of each sample were injected onto a C18 Pursuit 5 (50 mm × 2.0 mm) column (Varian) coupled to a double mass spectrometer in tandem (Varian 320 MS-MS 3 ). The mobile phase comprised solvent A (0.05% formic acid) and solvent B (0.05% formic acid in methanol) as follows: 85% solvent A for 1 min 30 s (flow rate 0.4 ml/min), followed by 3 min in which solvent B increased till 98% (0.2 ml/min) which continued for 5 min 30 s with the same flow rate, followed by 2 min 30 s with increased flow rate (0.4 ml/min), subsequently returning to 85% solvent A in 1 min, conditions that were kept till the end of the run, in total 15 min. Compounds were detected in the electrospray ionization negative mode. Molecular ions [M-H] − at m/z 137 and 209 and 141 and 213 generated from endogenous SA and JA and their internal standards, respectively, were fragmented under 12 V collision energy. The ratios of ion intensities of their respective daughter ions, m/z 93 and 97 and m/z 59 and 63, were used to quantify endogenous SA and JA, respectively.
QUANTITATIVE RT-PCR
For analysis of transcript levels, total RNA was isolated using Trizol from 10 infiltrated leaves, harvested from 10 different plants, in three independent experiments and treated with TurBo DNAfree (Ambion 4 ) to remove DNA. cDNA was synthesized from 1 μg of total RNA using M-MuLV reverse transcriptase (Fermentas 5 ), as described by the manufacturer, in a 20-μl reaction that was diluted to 50 μl prior to using it for the real-time PCR. This was performed in a 20-μl volume containing 2 μl of cDNA, 0.4 pmol of specific primer sets for each gene and 10 μl of iTaq TM SYBR Green Supermix with ROX (Bio-Rad 6 ). PCR conditions were as follows: 95 • C for 2 min 30 s (first cycle), 95 • C for 15 s and 60 • C for 30 s (40 cycles). To ensure amplification of a single product during the qRT-PCR reactions, a dissociation protocol was performed in which samples were slowly heated from 55 to 95 • C. qRT-PCR was performed using the ABI Prism 7000 real-time PCR detection system (Applied Biosystems) and the data were collected using software (ABI 7000 SDS version 1) provided by the supplier. Transcript levels were normalized to the levels of the SAND gene (At2g28390; Hong et al., 2010) and quantification was performed as described in previous work (Pfaffl, 2001). Primer sequences were as reported in (Anderson and Badruzsaufari, 2004;Czechowski et al., 2005;Park et al., 2010) for PR1, VSP2, LEC, and SAND, respectively.
TRYPAN BLUE AND ANILINE BLUE STAINING
Trypan blue staining solution was prepared by adding trypan blue to lactophenol (10 ml lactic acid, 10 ml glycerol, 10 ml phenol, and 10 ml distilled water) to a concentration of 2.5 mg/ml. Two volumes of ethanol were added to the trypan blue-lactophenol solution. To visualize plant cell death, mock and DC3000 infected leaf tissues were placed in plates containing staining solution and heated in a microwave at intervals for 1 min. The plates were incubated for 2 h at room temperature, followed by destaining (three times) in chloral hydrate (2.5 g/ml). The leaf tissues were mounted in 70% glycerol for observations with a microscope. For detection of callose deposition, leaves were incubated for at least 24 h in 96% ethanol until all tissues were transparent and stained in 0.01% aniline blue in 0.15 M K 2 HPO 4 (pH 8.5). Leaf tissues were incubated for 1.5-3 h, mounted on slides, and observed under an epifluorescence microscope (AF6000) with UV filter (excitation filter: BP 470/40 nm; emission filter: BP 525/50 nm).
CALLOSE QUANTIFICATION
Callose was quantified from digital photographs as the number of white pixels, covering the whole leaf material, using Photoshop CS7 software. Contrast settings of photographs were adjusted to obtain an optimal separation of the callose signal from the background signal. Callose was selected automatically, using the "Color Range" tool. In cases in which the contrast settings resulted in significant loss of callose signal, due to high autofluorescence of vasculature tissue, callose was selected manually, using the "Magic Wand" tool of Photoshop CS7. Relative callose intensities were quantified as the number of fluorescent callose-corresponding pixels relative to the total number of pixels covering plant material (Luna et al., 2011).
E -2-HEXENAL TREATMENT
Plants were grown for 3 weeks under the conditions mentioned above before being exposed to volatiles. For the volatile treatment, 10 plants in single pots were placed into airtight glass desiccators (22 l). E-2-hexenal was diluted in methanol, and applied to a sterile cotton swab, placed in an Erlenmeyer flask, between the plants in the desiccators to give a final concentration of 3 μM. For the control treatment, only methanol was applied. Plants were incubated in the desiccators for 24 h and subsequently taken out to be placed under the growth conditions described above for 1 h, prior to infiltration with bacteria or mock solution as mentioned above. E-2-hexenal was purchased from Sigma-Aldrich.
hpl1 INFLUENCES SUSCEPTIBILITY TO Pseudomonas syringae pv. tomato (DC3000)
In order to determine whether the ability to synthesize GLVs had an effect on Arabidopsis susceptibility to pathogenic bacteria, we compared Landsberg erecta (HPL, Ler) and an introgression line between Col-0 and Ler that can synthesize only trace amounts of GLVs, hpl1 (Shiojiri et al., 2012), for the susceptibility to Pseudomonas syringae pv. tomato DC3000. To ensure infection throughout the entire leaf, we used the syringe infiltration method since it overcomes stomatal defenses and maximizes the number of responding cells (de Torres Zabala et al., 2009), and bacterial populations were determined 72 hpi (hours post-infection). Figure 1 shows that DC3000 populations were lower in the hpl1 line. The difference measured in bacterial population between Ler and hpl1 (∼4.6-fold) was statistically significant (t-test P < 0.05). This indicates that the hpl1 line is less susceptible to DC3000 than Ler. It is well known that the balance between JA and SA is crucial for the interaction that will be established between a pathogen and its host (Spoel and Dong, 2008;Grant and Jones, 2009;Pieterse et al., 2009). We therefore monitored the changes in JA and SA in Ler and the hpl1 plants, prior to the bacterial population measurement, at 2, 24, and 48 hpi. As shown in Figure 2A, the levels of JA were up at 2 hpi in all treatments, most likely because of the mechanical damage caused by the inoculation with the syringe. At 24 hpi, this wound response was reset, as JA levels were very low, comparable to the mock inoculation. The situation changed at 48 hpi when JA levels increased in DC3000 infested leaves, in Ler approximately threefold higher than in hpl1. SA levels ( Figure 2B) changed already at 24 hpi, with levels being approximately 1.7-fold higher in hpl1 than in Ler, suggesting that SA-related defenses are activated earlier in hpl1. In Ler, the SA levels were higher than in hpl1 at 48 hpi suggesting that these defenses are mounted later in Ler.
JA MARKER GENES ARE LESS INDUCED IN hpl1 THAN Ler WHEN INFECTED WITH DC3000
In order to determine whether the differences in hormone levels had an effect on the expression of relevant marker genes in our system, we performed qRT-PCR for genes downstream of JA and SA. We chose VSP2 and LEC for JA (Potter et al., 1993;Penninckx et al., 1998;Thomma et al., 1998;Liu et al., 2005;Pré et al., 2008) and PR-1 for SA (Bowling et al., 1997;Clarke et al., 2001). PR1 expression was clearly induced by DC3000 at 48 hpi, however, to similar levels in Ler and hpl1 plants (Figure A1 in Appendix). In contrast, transcript levels of both VSP2 and LEC at 48 hpi (and 24 hpi) were much lower in hpl1 than in Ler (Figures 3A,B). This result is consistent with the observed lower JA levels in hpl1 at 48 hpi (Figure 2A).
Ler (HPL) AND hpl1 DIFFER IN THE NUMBER OF DEAD CELLS AND IN CALLOSE DEPOSITION
To investigate further the differences between Ler and hpl1 in mounting plant defense responses, we decided to look at the appearance of dead cells and callose deposition. Dead cells are indicative of programed cell death (or the hypersensitive response, HR) and enhanced resistance, usually occurring when an pathogenic effector is recognized by the host (Alfano and Collmer, 1996), whereas callose is typically triggered by conserved pathogen-associated molecular patterns (PAMPs), such as flagellin, at the sites of infection during the relatively early stages of pathogen invasion (Brown et al., 1998;Gómez-Gómez et al., 1999;Jones and Dangl, 2006). Dead cells appeared earlier and more frequently in the more resistant hpl1 while callose deposition occurred earlier and more abundantly in the more susceptible Ler (Figures 4A-C). Dead cells appeared at day 2 in hpl1, whereas in Ler they were not present at all, even at day 3. Ler started to deposit callose massively at day 1, while much less papillae at this time could be observed in hpl1. Moreover, even at later stages of infection, at days 2 and 3, Ler showed more callose deposition than hpl1.
E -2-HEXENAL TREATMENT INCREASES SUSCEPTIBILITY TO DC3000
Since hpl1 is unable to produce GLVs, we addressed the question whether application of GLVs would restore its susceptibility to DC3000 comparable to Ler. We chose to use the C6-aldehyde E-2-hexenal, one of the most active GLVs, and treated hpl1 and Ler plants with 3 μM aerial E-2-hexenal or with the carrier methanol (MeOH) for the control treatment. Figure 5A shows that the treatment with the C 6 -aldehyde turned both hpl1 and Ler more susceptible to DC3000, as bacterial populations increased about five-and ninefold, respectively, in the E-2-hexenal pre-treated
FIGURE 3 | JA-dependent gene expression is higher in infected Ler plants. (A) VSP2 transcript levels and (B)
LEC transcript levels were measured by qRT-PCR in Ler and hpl1 infected with DC3000 at 24 and 48 hpi and normalized for SAND transcript levels. Bars represent the ratio between the transcript levels in infected and mock samples. Three infected or mock infiltrated leaves were harvested from three different plants and pooled for RNA isolation. Bars represent the mean of three independent experiments. Error bars represent standard error. Bars annotated with asterisk indicate significant differences among samples (P < 0.05, according to t -test analysis). leaves compared to the control pre-treatment ( Figure 5B). Additionally, we measured JA and SA levels in Ler and hpl1 plants infected with DC3000 after pre-treatment with E-2-hexenal or MeOH. Although JA and SA levels increased 48 hpi after DC3000 infection, no significant differences in hormone levels were detected between the E-2-hexenal and the control treatment or between Ler and hpl1 (Figure A2 in Appendix).
THE EFFECT OF E -2-HEXENAL ON BACTERIAL GROWTH ACTS VIA ORA59.
Since a functional HPL leads to higher susceptibility and higher JA levels upon DC3000 infection and E-2-hexenal pre-treatment increased susceptibility of Arabidopsis to DC3000 we sought to elucidate part of the signaling pathways involved, by testing if Arabidopsis mutants in the JA-signaling pathway were still more susceptible to DC3000 after treatment with E-2-hexenal. We chose to analyze MYC2 and ORA59 impaired lines since these are the main players in regulating JA-dependent responses and are located in two different branches of the JA-signaling pathway (Lorenzo et al., 2003(Lorenzo et al., , 2004Anderson and Badruzsaufari, 2004;Dombrecht et al., 2007;Oñate-Sánchez et al., 2007;Kazan and Manners, 2008; www.frontiersin.org The data presented are from a representative experiment that was repeated four times with similar results. All pre-treatments with E -2-hexenal were significantly different from the control treatment (P < 0.05, according to Student's t -test analysis). (B) Bars represent the ratio between cfu/cm 2 with E -2-hexenal pre-treatment and cfu/cm 2 with methanol pre-treatment (control). Values are the mean of three independent experiments. Error bars represent standard error. Pré et al., 2008). As shown in Figure 6A, myc2 (jin1-7) plants were more resistant to DC3000 as has been reported (Fernández-Calvo et al., 2011). Moreover, myc2 as well as wild-type plants showed increased susceptibility to DC3000 when pre-treated with E-2-hexenal, seemingly excluding a role for MYC2 in mediating this phenomenon. In contrast, the same assay performed on RNAi-ORA59 plants (Pré et al., 2008) showed that the bacterial populations increased significantly less in the ORA59 silenced plants compared to the corresponding control line after E-2hexenal treatment (Figure 6B). This indicates an involvement of ORA59 in this response to E-2-hexenal.
THE E -2-HEXENAL EFFECT IS CORONATINE DEPENDENT
Pseudomonas syringae pv. tomato strain DC3000 synthesizes COR (Mitchell, 1982), a phytotoxin that mimics JA-Ile (Thines et al., 2007;Yan et al., 2009), in order to antagonize the SA-dependent defenses (Brooks et al., 2005;Glazebrook, 2005). Therefore, we also determined whether the production of COR was necessary for DC3000 to proliferate more in E-2-hexenal treated plants. For this, Ler and hpl1 plants were infected with the Pseudomonas syringae mutant strain DC3682 (Ma et al., 1991), that is unable to produce COR, after pre-treatment with E-2-hexenal or methanol. shows that the bacterial populations of the cor mutant were only slightly, but significantly, higher in Ler or hpl1 plants treated with E-2-hexenal compared to the control plants, but that this increase was much lower than for DC3000 (Figure 1). Thus COR seems to be necessary for DC3000 to benefit from the E-2-hexenal treatment.
DISCUSSION
Green leaf volatiles have received considerable attention for their ability to induce direct and indirect defense responses in plants and can be considered important players in the already complex network regulated during biotic stress. However the mechanisms by which GLVs influence pathogenesis, and the signaling pathways involved in these responses, are not well known. To address this, we used Ler and its Arabidopsis introgression line, hpl1, lacking GLV synthesis, and analyzed their response during infection with the bacterial pathogen Pseudomonas syringae pv. tomato (DC3000). DC3000 was chosen because in some plant species such as lima bean and tobacco, infection triggers E-2-hexenal emission (Croft , 1993;Heiden et al., 2003). We hypothesized that hpl1 plants would be more susceptible to DC3000 since there is evidence that GLVs and E-2-hexenal have antimicrobial properties (Prost et al., 2005), induce defense-related genes or biosynthesis of defenserelated secondary metabolites (Bate and Rothstein, 1998;Arimura et al., 2001;Gomi et al., 2003;Weber et al., 2004;Farag et al., 2005;Kishimoto et al., 2005Kishimoto et al., , 2006Paschold et al., 2006), and increase resistance against B. cinerea (Kishimoto et al., 2005). However, we found the opposite result: plants impaired in GLV production were more resistant to DC3000 (Figure 1). A similar result was very recently shown in rice where the mutant Oshpl3, not able to synthesize GLVs, was more resistant to Xanthomonas oryzae pv. oryzae (Tong et al., 2012).
Subsequently, we investigated some of the mechanisms underlying this result by analyzing the levels of SA and JA since it is well known that these phytohormones and their antagonism are crucial for the development of pathogenesis in Arabidopsis (Spoel and Dong, 2008;Grant and Jones, 2009;Pieterse et al., 2009). Hormone measurements clearly showed that JA levels were much lower in hpl1 than in Ler (Figure 2A). Conversely, hpl1 showed an earlier induction of SA than Ler ( Figure 2B). These data suggest that a non-functional HPL gene influences the JA-branch of the oxylipin pathway, leading to lower production of JA when Arabidopsis is challenged with Pseudomonas. Thus, this is not related to substrate competition as previously shown in Arabidopsis where ectopic expression of HPL led to lower JA levels upon wounding (Chehab et al., 2006). Reduction of HPL expression in rice and N. attenuata also influenced JA levels but differently: Oshpl3 and asHPL1 had increased JA levels (Halitschke et al., 2004;Tong et al., 2012), in N. attenuata probably due to crosstalk between the GLV and JA pathway (Allmann et al., 2010).
Since JA-signaling downstream of COI1 occurs via two different branches, regulated by MYC2 or ORA59, we used markers for both branches to study their activation after DC3000 infection. LEC, a lectin-like gene, was used for the ORA59 pathway since it is induced by methyl-jasmonate and upon ORA59 overexpression (Schenk et al., 2000;Pré et al., 2008), while VSP2 was used for the MYC2 pathway (Abe et al., 2003;Dombrecht et al., 2007). Both VSP2 and LEC transcript levels were much lower in hpl1 than in Ler (Figures 3A,B) concurrent with the lower JA levels. Thus DC3000 activates in Ler, with an active HPL unlike Col-0 (Duan et al., 2005), with which most DC3000 experiments are carried out, both branches of the JA-signaling pathway and antagonistic control of these distinct branches of the JA pathway (Verhage et al., 2011) is apparently minor. Transcript levels of the SA-marker PR-1 were higher upon DC3000 infection, similarly in hpl1 and Ler (Figure A1 in Appendix), probably because the differences in SA levels between the two genotypes were not big enough to cause a difference. Thus it seems that the lower JA levels in hpl1 plants leads to less activation of the JA-signaling pathways and renders them less susceptible to DC3000.
A hallmark of basal plant defenses to pathogen infection is the deposition of callose. PAMP-induced callose deposition has recently been defined with essential roles for the DC3000 type III effector HopM1 and COR suppressing callose deposition, the latter being, interestingly, partly COI1-independent (Geng et al., 2012). Our results showed that in hpl1, although with smaller bacterial populations than in Ler, clearly less callose was deposited (Figures 4B,C). Ethylene (ET) signaling it is crucial for callose deposition in response to flagellin (Clay et al., 2009). It is possible that this ET signaling is less activated in hpl1, leading to less callose deposition. Support for this comes from our complementation studies with the hpl1 mutant, a response that is largely dependent on ORA59, a TF that integrates JA and ET signaling ( Figure 6B). Perhaps related to this is the fact that DC3000 is apparently less effective in preventing cell death in hpl1 than in Ler ( Figure 4A), with fewer living cells producing less callose. DC3000 apparently triggers in hpl1 a higher rate of cell death, which is related to higher resistance (Jones and Dangl, 2006).
With the aim to overcome the hpl1 phenotype in response to DC3000 infection, we decided to treat these, and Ler, plants with E-2-hexenal. The pre-treatment with 3 μM E-2-hexenal for 24 h prior to DC3000 infection made hpl1 plants considerably more susceptible to DC3000 (Figures 5A,B). The increase in bacterial populations was about ninefold in Ler and fivefold in hpl1 plants. Thus Ler plants remained more susceptible to DC3000 than hpl1 plants, most likely due to the functional HPL. Due to its high reactivity for being a reactive electrophile species (RES), E-2-hexenal, either induced during the HR or exogenously applied, can undergo conjugation to glutathione (GSH), leading to the formation of E-2hexenal-GSH adducts in the form of 1-hexanol-3-GSH (Davoine et al., 2006;Mirabella et al., 2008). Conjugation to GSH is a well-known mechanism to inactivate reactive molecules (Coleman et al., 1997). Additionally, conjugation to cellular proteins has been reported to occur for several RES, including E-2-hexenal (Davoine et al., 2006;Myung et al., 2007;Dueckershoff et al., 2008;Mueller et al., 2008;Yamauchi et al., 2008). Therefore, we cannot exclude the possibility that, through conjugation, E-2-hexenal affects the function of proteins involved in the plant defense responses to DC3000, making Arabidopsis more susceptible to this pathogen. A similar effect has been reported for syringolin, a toxin with an unsaturated α,β carbonyl moiety, that makes it a RES, produced by, e.g., Pseudomonas syringae pv. syringae. This toxin specifically inhibits the proteasome in order to suppress host defenses (Groll et al., 2008;Schellenberg et al., 2010).
Analyses of phytohormone levels after treatment of E-2hexenal and DC3000 infection showed that there were no statistically significant differences in SA and JA levels between control and treatment (Figure A2 in Appendix). So far only in monocots (maize) an increase in JA has been measured after a GLV treatment (Engelberth et al., 2004;Engelberth, 2011). In the JA-signaling pathway COI1 plays a central role and mutants in this gene are blocked in almost all JA responses (Feng et al., 2003;Devoto et al., 2005;Wang et al., 2008). Downstream of COI1, different TFs regulate specific JA-dependent responses: MYC2 and ORA59 are the main players involved. The MYC2-dependent branch is associated with wound response, responses against herbivores and is also regulated by abscisic acid (ABA; Lorenzo et al., 2003). This basic helix-loop-helix (bHLH) transcription factor regulates a large number of JA-responsive genes (Dombrecht et al., 2007), among which VEGETATIVE STORAGE PROTEIN2 (VSP2; Liu et al., 2005). In the other branch, ORA59 integrates JA and ET signaling (Pré et al., 2008). Interestingly, in spite of the absence of difference in JA and SA levels, the higher susceptibility of Arabidopsis plants to DC3000 after E-2-hexenal treatment was dependent on ORA59. The DC3000 bacterial populations increased only slightly in ir-ORA59 plants after E-2-hexenal treatment as compared to control (35S-GUS) plants (Figure 6B), indicating the relevance of JA signaling, and perhaps ET signaling. A role for MYC2 in this process was excluded based on the fact that myc2 mutants still responded to exogenous E-2-hexenal treatment ( Figure 6A).
From the bacterial side we investigated whether the production of COR was necessary to benefit from the E-2-hexenal treatment. For this we employed cor, a COR-deficient strain, to infect plants, after the E-2-hexenal or control treatment. The result showed that there was a small but significant increase in bacterial populations of the cor strain after the E-2-hexenal treatment (Figure 7). Nevertheless this difference was much smaller than for DC3000, suggesting that COR is necessary for DC3000 to fully benefit from GLVs.
Our data show that a functional HPL in Arabidopsis promotes susceptibility to DC3000. This effect is partially mediated by ORA59 in the plant and by COR in the bacteria.
The question remains how DC3000 precisely exploits HPL or its products, GLVs or the C 12 compounds that are also formed in the HPL pathway (Kallenbach et al., 2011), for its benefit. Since it is clear that some herbivores can lower HPL transcript levels (Halitschke et al., 2004;Savchenko et al., 2012), we propose that HPL may be a target for DC3000 to employ in Arabidopsis, albeit to its own advantage. | 2016-05-12T22:15:10.714Z | 2013-04-12T00:00:00.000 | {
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228846466 | pes2o/s2orc | v3-fos-license | The Use of Finite Mixture Models and EM-Algorithm to Analyze Grain Structure in HPT-Nanostructured Metallic Materials
Analysis of grain size distribution’s histograms of Nb and Ni subjected to high-pressure torsion at cryogenic temperatures and Nb3Sn layers formed in Nb/Cu – Sn composite wires after the diffusion annealing has been carried out using statistical analysis method based on the application of finite mixture models and using an expectation – maximization algorithm with the estimation of fitting accuracy by the Bayesian information criterion. It has been established that the approximation by the model with a single component of logarithmic standard distribution is the most suitable for all examined experimental distributions in contrast to the model with two components. Besides, the use of the proposed approach allows to practically eliminate an influence of the additional errors in the experimental data which seem to be introduced at transmission electron microscopy image processing and constructing histograms of grain size distributions.
Introduction
In the last decades materials with ultrafine-grained (UFG) structure attract great attention of the researchers due to their unique physical and mechanical properties. UFG materials possess a grain size below 1000 nm and exhibit significantly higher strength, unusual mechanical behavior and a complex of unique properties in comparison to their coarse-grained counterparts [1][2][3].
The application of severe plastic deformation (SPD) enables obtaining bulk UFG materials. A number of various techniques of SPD have been developed by now [4], and one of the earliest and most common among them is high-pressure torsion (HPT) [5,6]. This method makes it possible to produce a structure close to nanocrystalline [7][8][9][10][11][12][13][14] at room temperatures, and HPT at cryogenic temperatures allowed the authors of [15][16][17][18][19] to obtain a true nanocrystalline structure in pure metals.
It was shown [20][21][22] that grain boundaries as an element of structure playing a determinative role in UFG materials markedly differ from those in coarse-grained polycrystals and are referred to as "nonequilibrium" or deformation-modified boundaries. Besides, such grain boundaries are characterized by high density of defects and, accordingly, enhanced energy.
Since the grain structure is the most important factor responsible for the special properties of such materials, in the studies devoted to investigations of structure in nanostructured materials [7][8][9][10][13][14][15][16]18,19] transmission electron microscopy (TEM) with a subsequent processing of TEM images is used. However, in a lot of these studies, the statistical analysis of structure usually deals with the grain sizes distributions described by a logarithmic standard (normal) distribution. At the same time, it was shown [23] that the histograms of the grain size distribution in the nanostructured materials both of the HPT and of the nanostructures of recrystallization origin are not always described only by the logarithmic standard distribution. Besides, the different models can be used to fit the experimental distributions. That may not lead to the same results because of different accuracy of fitting techniques and models [24,25]. Since experimental distributions may be a mixture of several log-normal distributions with different values of the mean, standard deviation and volume fraction, a small amount of one of distributions may be estimated as contamination. Moreover, a logarithmic normal distribution is not symmetrical that additionally complicates the process of fitting.
Therefore, the aim of this work is to propose a robust statistical approach to analyze experimental grain sizes distributions.
Results and discussion
The results of studies [15,18] devoted to nanostructuring of pure metals by HPT at cryogenic temperatures had been used. It is assumed that an effect of recrystallization both during deformation and after that was completely eliminated. The data on the nanostructure formed during the annealing of highly deformed composites is used [26] as well.
The analysis of grain sizes distributions was performed in terms of that the experimental distributions of grains are described by lognormal function [27]. Pearson's mixture model-based approach known as finite mixture models was chosen to describe the experimental distributions [26]. An expectationmaximization (EM) algorithm [28,29] has been used for fitting the experimental distributions.
In addition, it is suggested that the experimental distribution may be both single distribution and a sum of separated logarithmic standard distributions, so the expression of the model is as follows: where μi and σi are the mean and the standard deviation of the variable of the natural logarithm distribution; Ai is a scale factor, which is the estimation of volume fraction of the grain group in the experimental distribution; i -grain group number; N -the amount of distributions in the model. EM-algorithm is common technique used to determine the parameters of a mixture. At the same time, EM-algorithm is of particular appeal for finite normal mixtures with an a priori given number of components [28,29]. Therefore, in order to use EM-algorithm with the most efficiently it is necessary to describe experimental distribution as normal distribution's mixture.
Since, a log-normal distribution is a continuous probability distribution of a random variable whose logarithm is normally distributed, the model (1) can be represented in logarithmic scale as: Due to μi and σi have logarithmic scale, these parameters should be turned into normal scale by follows expressions: where Mi and Si are the mean and the standard deviation of the standard distribution in the normal scale, respectively. It should be noted that the approach assumes that initial data for processing is a raw set of grain sizes. In order to process experimental grain sizes distributions, the distributions should be decomposed into a raw set of grain sizes in contrast to fitting of distributions by curves in accordance with the technique of work [23][24][25]. The next step of processing is to represent the set of grain sizes on the logarithmic scale. Then the data was processed by EM-algorithm with different the amount of distributions (N) in accordance with the model. The advantage of the technique is the use of EM-algorithm with enough amount of random start points to avoid incorrect results. Moreover, modern implementations of EMalgorithm for fitting of a Gaussian mixture model allow to compute the Bayesian information criterion (BIC) [30] to assess the number of clusters in the data. That can be used to select the number of components (N) in a model in an efficient way.
In Table 1, the results of the calculation are summarized, as well. It can be seen that the approximation by the model with a single component (N = 1), in accordance with the calculated BIC values, is the most suitable for both the grain structure of Nb and Ni subjected to HPT at cryogenic temperatures and Nb3Sn structure (Composite) formed in Nb/Cu-Sn composite wires after the diffusion annealing in contrast to the model with two components (N = 2). In addition, the data processing results of experimental grain sizes histograms from works [15,18,26] with using the most suitable model are represented in Figure 1 where the experimental histograms and calculated curves in both normal and logarithmic scale are shown.
Earlier [23] it was shown that the fitting of the experimental grain sizes histograms by only single log-normal distribution gives significant imprecision for all these materials. Whereas, use of additional normal distribution in the fitting model allowed to describe the experimental data with much greater accuracy in accordance with the calculated chi-square values. Besides, the fraction of the additionally introduced standard distribution in all cases was considerable and even exceeded the fraction of the logarithmic distribution in the case of Nb.
It can be concluded that the common technique based on the analysis of TEM image with a subsequent processing and constructing histograms of grain size distribution introduce additional normally distribution errors. This fact seems to be the main reason for appearing the additional normal distribution in experimental histograms. At the same time, the use of statistical analysis to describe the experimental distributions in terms of finite mixture models with using EM-algorithm allowed to practically eliminate an influence of the additional errors in the experimental data. [15,18,26] and approximation results.
Summary
The analysis of grain sizes distributions was performed in terms of that the experimental distributions of grains are described by the lognormal function. Pearson's mixture model-based was chosen to describe the experimental distributions. An expectation-maximization algorithm has been used for fitting the experimental distributions of the grain structure of Nb and Ni subjected to HPT at cryogenic temperatures and Nb3Sn structure formed in Nb/Cu-Sn composite wires after the diffusion annealing.
The analysis of fitting accuracy results was shown that the approximation by the model with a single component is the most suitable for all examined experimental distributions in contrast to the model with two components. Besides, the use of the proposed approach allows to practically eliminate an influence of the additional errors in the experimental data which are introduced at TEM image processing and constructing histograms of grain size distributions. | 2020-11-19T09:15:33.352Z | 2020-11-13T00:00:00.000 | {
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8058660 | pes2o/s2orc | v3-fos-license | Diagnostic value of medical thoracoscopy in malignant pleural effusion
Background Medical thoracoscopy has been shown to be an efficacious procedure in diagnosing unexplained exudative pleural effusions with excellent safety. This study aimed to assess the diagnostic significance of thoracoscopy in the management of patients with malignant pleural effusion (MPE). Methods Consecutive patients with malignant pleural effusion were retrospectively reviewed, and their demographic, radiographic, thoracoscopic and histological data were collected. Results Between July 2005 and June 2014, 342 of 833 patients undergoing thoracoscopy were finally confirmed to suffer from MPE. The top three frequent causes of MPE were metastatic carcinoma (79.5%), malignant mesothelioma (10.2%), and lymphoma (2.9%). Among metastatic malignancies, the most common cancer was lung cancer (85.2%), followed by breast cancer (4.4%), ovarian cancer (2.2%), pancreatic cancer (1.8%), etc. No serious adverse events associated with thoracoscopy were recorded. Conclusions Medical thoracoscopy is a valuable and safe tool in diagnosing malignant pleural effusion with minimal complication rates.
Background
The identification of malignant cells in a pleural lavage in patients without pleural effusion suggests micrometastatic disease, and our previous meta-analysis [1] showed that positive pleural lavage cytological findings are associated with a higher recurrence rate and significant poorer survival, with the overall hazard ratio for patients having malignant cells in pleural lavage was 5.61 (95% confidence interval 3.98-7.90). In non-small-cell lung cancer patients, the evidence of even a minimal pleural effusion at diagnosis is an independent prognostic factor for worse survival [2]. Malignant pleural effusion (MPE) is frequently observed in multiple malignancies, and lung cancer is the most common cause [3]. The existence of MPE in patients indicates systemic dissemination of cancer and declining in life expectancy and quality [4,5].
The current guideline recommended that thoracentesis and/or closed pleural biopsy can be used as the first diagnostic steps in the diagnosis of MPE [6]. However, these procedures usually do not work when pleural effusion with thickness less than 10 mm on chest computed tomography (CT) scans. Instead, the more invasive approaches, such as medical thoracoscopy (MT), can be considered to identify whether pleural biopsy contains malignant cells [3,7]. As a matter of fact, MT is a highly sensitive and safe method for diagnosing exudative pleural effusions [8][9][10]. The recent developed semirigid MT is easy to use and can gain popularity among respiratory physicians who are accustomed to flexible bronchoscope [11,12].
In the present retrospective study of patients with MPE having undergone at least one semi-rigid MT over a 9-year period in a Chinese 1600-bed general hospital, we analyzed the diagnostic efficiency and safety of MT in the diagnosis of MPE.
Methods
The study protocol and ethical approval was approved by the Institutional Review Board for human studies of Beijing Chao-Yang Hospital, China. Informed consents were not required as this was considered a review of clinical practice.
Information including medical history, clinical presentation, laboratory examination results, and image data of unexplained exudative pleural effusions patients who underwent MT in our hospital between July 2005 and June 2014 were gathered, and only MPE patients were finally included in the current study. Unexplained exudative pleural effusions were defined as the patients underwent the initial diagnostic approaches including thoracentesis and/or closed pleural biopsy, and their diseases remain undiagnosed. The characteristics of the study population are listed in Table 1.
MT procedures have been described in our previous publications [13,14]. The diagnosis of MPE was established by the presence of the positive findings for malignancy in pleural biopsy.
Descriptive statistical methods were used in the data analysis (mean ± standard deviation [SD] or/and range).
Results
Between July 2005 and June 2014, 833 patients with undiagnosed pleural effusions successfully underwent medical thoracoscopy [15]. Eventually, 342 patients with lymphocytic exudates were finally diagnosed with MPE; the mean age was 62.8 ± 9.7 years.
For 149 MPE patients, pleural fluid occurred only in the right side, for 133 only in the left, and for the rest 60 both sides were involved ( Table 1). The size of a pleural effusion was clarified as small, moderate, or large based on CT imaging according to the methods described by Moy and colleagues [16]. In both unilateral and bilateral effusion, the proportions of small, moderate, and large size of pleural effusions were 16.7, 12.9, and 70.4%, respectively. The appearance of pleural effusion was blood-stained in 55.9% of patients, and in 44.1% was yellow.
In addition to pleural effusion, CT imaging revealed mediastinal and hilum lymphadenopathy, pleural thickening, pulmonary consolidation or infiltration, pulmonary mass or nodules, pulmonary atelectasis, and pleural nodularity (Table 1).
In all patients studied, we observed one or more abnormalities on the surface of parietal or/and visceral pleura under medical thoracoscopy. As shown in Table 2, pleural nodules, hyperemia, pleural adhesion, pleural plaques, ulcer, and the other pleural pathological changes were observed.
The most common etiological causes of MPE were metastatic carcinomas (n = 272), pleural malignant mesothelioma (n = 35), lymphoma (n = 10). It should be mentioned that we could not identify the original malignancies in 25 patients with MPE (Table 3). Among metastatic malignancies that resulted in MPE, the most common cancer included lung cancer, followed by breast cancer, ovarian cancer, pancreatic cancer (Table 4).
No serious adverse events were observed, and transient chest pain (43.9%) induced by the indwelling chest tube was the most frequent minor complication. Subcutaneous Other pleural pathological changes 97 (28.4) emphysema was found in 8.5% of patients who recovered after chest-tube drainage. Minor bleeding was recorded in 6.4% of patients. And 5.6% of patients appeared with transient self-limited fever (38°C or more).
Discussion
Because the prognosis for patients with MPE is poor, an efficacious procedure that can establish a definite diagnosis as early as possible with a minimum of risk and discomfort would be highly desirable. If a patient with undiagnosed pleural effusion is suspected as malignant, cytologic examination of pleural fluid is the first recommendation [6]. Although repeated thoracenteses can enhance the sensitivity of cytology, it is usually only 50 to 70% [17]. When cytology fails, closed percutaneous needle biopsy was traditionally performed blindly by an Abrams or Ramel needle [18][19][20]. Nevertheless, its role in diagnosing MPE has been challenged, as the positive diagnostic rate of closed pleural biopsy was only about 50% [21,22]. More recently, the real time image-guided pleural biopsy has been shown to be a promising procedure for sampling the pleura, since it can increase the sensitivity for diagnosing MPE to about 80% [21,[23][24][25]. Numerous tumor markers have been intensively examined for improving the diagnosis of MPE, however, seeking for a highly accurate pleural fluid tumor marker that reliably diagnoses MPE has been in vain so far [26]. Using one tumor marker alone for diagnosing MPE is not recommend according to the recent evidences, however, when combined two or more tumor markers together, the diagnostic sensitivity seems to be improved [27,28]. The diagnostic performance of tumor markers for MPE seems to be similar with conventional tests including cytological examination-high specificity and low sensitivity. Tumor markers are less important in practice, since they do not complement the properties of conventional tests.
CT-or ultrasound-guided pleural biopsies are quite sensitive and safe, with the only reported complications being local hematoma and minor hemoptysis [21,29]. The limitation of the image-guided pleural biopsy is the blindness of the procedure. MT overcomes this problem by allowing for the visualization of abnormal areas and for a direct biopsy, and thus improves the diagnostic accuracy of pleural effusions [8,9]. Since June 2005, our institution started using MT as a routine method for patients with undiagnosed exudative pleural effusion in cases when either clinical, radiologic, laboratory, or cytologic investigation was failed. During a period of 9 years, 833 patients with unexplained pleural effusions underwent MT successfully, and among them, 342 were eventually diagnosed with MPE [15].
One or more abnormalities on the surface of parietal or/ and visceral pleura were observed in the whole population in this study, including pleural nodules, hyperemia, pleural adhesion, pleural plaques, ulcer, and the other pleural pathological changes. Pathological examination revealed the presence of the positive findings for malignancy in pleural biopsy in 342 patients. The data in detail presented in the current study derived from our whole MT study population [15]. As reported in the previous publication [15], after a complete work-up including MT biopsies, the definite diagnoses of 92.6% (771/833) of patients with pleural effusions can be established definitely by MT followed by histopathological study.
It was noted that no etiological causes of pleural effusions can be identified in 7.4% (62/833) of patients even after MT [15]. All of these patients were followed up for [30][31][32]. Therefore, we cannot exclude the possibility that a few patients with MPE-negative MT results would be finally diagnosed with MPE if we prolong the follow-up. It has been reported that carcinoma from any organ can metastasize to the pleura, but lung, and breast carcinomas and lymphomas are the most common causes, digestive and ovary carcinomas are less frequent [33]. Our recent unpublished data indicated that during the past 3 years, 23.7% (365/1541) of pleural effusion patients admitted to our hospital were diagnosed with MPE. In the present study, our data showed that the most common etiological causes of MPE confirmed by MT were metastatic carcinomas, and followed by pleural malignant mesothelioma, lymphoma, and the other malignancies. Among metastatic malignancies that resulted in MPE, the most common cancer was lung cancer, followed by breast cancer, ovarian cancer, and pancreatic cancer.
MT has an excellent safety profile when performed by a trained physician, and a mortality rate associated with MT is always ≤0.8% [34]. A recent meta-analysis further suggested that mortality associated with MT was not observed, and that the major complication rate of MT was 1.5% and the minor complication rate was 10.5% [10]. In our 9-year study, MT procedures were well tolerated with a low rate of complications without serious adverse events, and transient chest pain caused by the indwelling chest tube was the most frequent minor complication. Large volumes of pleural fluid could be safely aspirated, although some patients suffered from coughing and chest discomfort after lung re-expansion with a chest tube.
The strengthen of this study was that study population was in a large size, in which 342 MPE patients were included. At the same time, our study also had limitations. First, as a retrospective study, it's impossible to collect and analyze the required data in a prospective way. Second, we could only retrospectively reviewed the data from patients with MPE, and no data from the other control group, such as the patients with tuberculous pleurisy, were available, it was therefore not possible to calculate the sensitivity and specificity of MT in diagnosing MPE. Third, blind needle biopsies or image-assisted biopsies were performed only in a few patients before undergoing MT in our series. This partially explained why there were so many pleural effusion patients (833 cases) receiving MT examination in our institution during 9 years.
Conclusions
In summary, MT is simple and safe with a high positive rate in the diagnosis of MPE. Due to its convenience and compatibility with existing bronchoscopy, MT appears to be a more widely performed procedure. Thus MT should be performed actively for proper patients with suspected MPE. | 2017-08-08T19:37:03.851Z | 2017-08-04T00:00:00.000 | {
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244921111 | pes2o/s2orc | v3-fos-license | Dynamics of two dark solitons in a polariton condensate under non-resonant pumping
We theoretically investigate the dynamics of two dark solitons in a polariton condensate under nonresonant pumping, based on driven dissipative Gross-Pitaevskii equations coupled to the rate equation. In particular, the analytical expression of the effective potential between two dark solitons is given. The resulting equation of motion captures how the open-dissipative character of a polariton Bose-Einstein condensate affects the properties of dynamics of two-dark soliton, i.e., two-dark soliton relax by blending with the background at a finite time. We further simulate the relative motion of two dark solitons numerically with the emphasis on how two solitons' motion being manipulated the initial velocity, which are in excellent agreement with the analytical results. The prediction of this work is sufficient for the experimental observations within current facilities.
We theoretically investigate the dynamics of two dark solitons in a polariton condensate under nonresonant pumping, based on driven dissipative Gross-Pitaevskii equations coupled to the rate equation. The equation of motion of the relative center position of two-dark soliton is obtained analytically by using the Lagrangian approach. In particular, the analytical expression of the effective potential between two dark solitons is given. The resulting equation of motion captures how the open-dissipative character of a polariton Bose-Einstein condensate affects the properties of dynamics of two-dark soliton, i.e., two-dark soliton relax by blending with the background at a finite time. We further simulate the relative motion of two dark solitons numerically with the emphasis on how two solitons' motion being manipulated the initial velocity, which are in excellent agreement with the analytical results. The prediction of this work is sufficient for the experimental observations within current facilities. Introduction.-At present, there are significant research interests in exciton-polariton Bose-Einstein condensates (BEC) in semiconductor microcavities as a novel platform for realization and investigation of nonlinear physics [1][2][3][4]. On the one hand, at the mean-field level, the static and dynamic properties of a polarition BEC can be well described by the nonlinear Schrödinger equation or Gross-Piteavskii equation (GPE), which has been a paradigm of theoretical and experimental studies of coherent nonlinear dynamics [5][6][7][8]. On the other hand, a polariton condensate is intrinsically non-equilibrium, with coherent and dissipative dynamics occurring on an equal footing. This has provided a new stage for practical applications of the GPE. Up to now, the nonequilibrium nature of the polariton has resulted in numerous intriguing nonlinear phenomena in a polariton condensates [9,10].
In the quest for novel scenarios that display combined effects of dissipation and nonlinearity on the nonlinear phenomena, the study of soliton in polariton condensates is among the hottest topics, with an emphasis on capturing the non-equilibrium nature of the soliton with no analog in the static counterpart [11][12][13][14][15][16][17][18][19][20]. Generally speaking, soliton is a kind of self-reinforcing solitary wave, which is formed by the cancellation of nonlinear effect and dispersion effect in medium and it can maintain its shape during propagation [21][22][23][24]. The dark or bright soliton can exist provided the interaction is repulsive or attractive [25,26]. In a polariton condensate, the nonlinearity of the polariton condensate arises from the strongly and repulsively interacting excitons, where the interaction can be controlled via Feshbach resonance [27,28]. A series of experiments have demonstrated the existence of the oblique dark solitons and vortices [29][30][31], or bright spatial and temporal solitons [32]. For example, in condensates created spontaneously un-der incoherently pumping, the formation and properties of dark solitons have been investigated theoretically in Ref. [33], and the existence of stable dark soliton trains has been reported in the non-resonantly driven spinor polariton BEC at one dimension [34]. However, to our best knowledge, previous studies on solitons in a polariton condensate have been limited to the one-soliton problem, whereas the two-soliton problem in non-equilibrium polariton BEC has remained so far unexplored. It is the purpose of the present work to investigate how the interplay of nonlinearity, dispersion and dissipation affects the existence and properties of two-dark soliton in a polariton BEC.
In this Letter, we present the first analytical result on the two-dark soliton problem in the context of a polariton BEC formed under non-resonant pumping by solving the dissipative GPE. First, we use the variational approach and analytically derive the time evolution equations for the soliton parameters, i.e., the relative distance between solitons. We compare this analytical result with the numerical solutions for the trajectory of two solitons directly obtained from the dissipative GPE, finding a remarkable agreement between the two. Our results open a new route to observe stable two-solitons in nonequilibrium polariton BEC within current experimental facilities.
Model.-We are interested in a exciton-polariton BEC under nonresonant pumping created in a wire-shaped microcavity similar to the one implemented in Ref. [35] that bounds the polaritons to a quasi-one-dimensional (1D) channel. Within the framework of the mean field theory, the polariton field described by the condensate order parameter of ψ(x, t) evolves along an effectively 1D driven-dissipative GPE coupled to a rate equation for the density n R (x, t) of reservoir polaritons as follows [36], arXiv:2112.03559v1 [cond-mat.mes-hall] 7 Dec 2021 In Eqs. (1) and (2), the m is the effective mass of lower polaritons and P is the rate of an off-resonant continuous-wave pumping; γ C and γ R describe the lifetime of the condensate and reservoir polaritons respectively, and R is the stimulated scattering rate of reservoir polaritons into the condensate; g C and g R characterize the strength of nonlinear interaction of polaritons and the interaction strength between reservoir and polariton respectively. Note that the parameters of g C , g R , and R have been rescaled into the one-dimensional case by the width d of the nanowire thickness as that . The emphasis and value of this work are to take account of the intrinsic dissipation and look for the possibility of the existence and dynamics of two-dark soliton in a polariton condensate. It's well known that the dark soliton is characterized with a localized dip in the condensate density with an associated phase gradient. Hence, we first need to determined the steady state of the model system based on Eqs. (1) and (2), which serves as the density background of the dark soliton's propagation. Directly following Ref. [36], the stable condensate appear under the condition of the pumping P in Eq. (2) being bigger than a critical value of P th = γ R γ C /R. In such, the steady homogeneous condensate and reservoir densities are expressed as follows: For convenience, we proceed to rescale Eqs. (1) and (2) into the dimensionless form. In more details, we rescale ψ → ψ/ n 0 C and introduce m R = n R − n 0 R ; as a result, Eqs. (1) and (2) can be rewritten as Here, the dimensionless parameters are given byḡ R = g R /g C ,γ C = γ CγR /γ R andR = R/g C n 0 C . Moreover, the time t and the space coordinate x are measured in the units of τ 0 = gn 0 C and x h = 2 /mgn 0 C . Note that Equations (3) and (4) are the starting point of investigating the two-dark soliton problem in the context of the polariton BEC. The non-equilibrium nature of model system is captured by the parameters ofR in the right hand of Eq. (3). Blow, we are theoretically interested in how the nonequilibrium nature affecting the dynamics of two-dark soliton.
Two-dark Soliton-Before investigating the effects of the non-equilibrium nature of model system characterized byR on the two-dark soliton solution, we first briefly review some important features of the normal GPE, corresponding to Eq. (3) withḡ R =R = 0. Under the boundary condition of ψ → 1 as |x| → ∞, the two-darksoliton solution can be written as [37] with A 2 + B 2 = 1. Here x ± = B (x ± x 0 ) is referred as to the center position of two dark solitions and 2x 0 can be treated as the relative center position between two solitons. In general, we can obtain that x 0 = v s t, A = v s and B = 1 − v 2 s with v s being the velocity of the dark soliton. In order to better understand the trial wavefunction of two dark solitons in Eq. (5), we have plotted the density profile with the parameter of v s = 0 in Fig. 1 (a) characterized that the phase of the two dark soliton solution ψ(x, t) has π phase jump profile (see Fig. 1
(b)).
Adding the open-dissipative effects as captured bȳ R introduces an external perturbation of the two-darksoliton in Eq. (5), which leads to two immediate consequences: first, all the parameters of two-dark-soliton solution in Eq. (5) become slow functions of time, i. e. A → A(t), B → B(t), and x 0 → x 0 (t), while the functional form of the soliton remains unchanged, which is at heart of the Lagange approach of quantum dynamics of two-dark soliton in the presence of perturbation; second, it's supposed that the two-dark-soliton will relax by blending with the background at a finite time. In such, we will rely on the Lagarange approach of the dark soliton perturbation theory which allows for the analytical treatment of the effects of the right hand in Eq. (3).
We focus on the relative center mass position of twodark-soliton solution corresponding to the time variation of the parameter of x 0 (t). As such, we can obtain the equation of motion of x 0 by employing the Lagrangian approach for the perturbation theory of soliton as Refs. [37][38][39], reading d dt dx being the Lagrangian and R * = (ḡ R − i 2R )m R being referred to as dissipative effects. The next calculations of the right-hand side of Equation (6) require knowledge of the reservoir density m R , In this work, we have limited the calculations in the fast reservoir limit, under which Next, directly following Ref. [38], we employ the variational method by plugging Eqs. (5) and (7) into Eq. (6) and obtain the equation of motion of the relative center motion of the two-soliton as follows: with V (x 0 ) = 2B 4 exp(−4x 0 B) being the effective potential of the relative center motion of the two-soliton and the dissipation-induced force F eff , reading Equation (8) is the key result of this work, which allows us to interpret the dynamics of the two-dark soliton in terms of the motion of a classical particle trapped in an effective potential V (x 0 ) subjected to an external force F eff . Equation (8) can recover the corresponding result in Ref. [38] as it's expected. From Eq. (8), it is clear that the key physics is that the effective potential of V (x 0 ) is repulsive; as a result, the two dark solitons is supposed to repel each other when they move close to each other. Based on Eq. (8), we conclude that the effective potential V (x 0 ) which decays exponentially with the x 0 dominates in the case of x 0 1, i.e., two dark solitons is relative closer to each other; in contrast, the dissipation effect captured by F eff plays a more important role is the case of x 0 1, resulting in that two-dark soliton relax by blending with the background at a finite time.
Above, we have developed the analytically physical picture of two-dark-soliton solution based on Eq. (8) and predicted features of the dissipation affecting the dynamics of two-dark soliton compared to the coherent case. In what follows, we plan to investigate dynamics of two-dark soliton solution in a more broader parameter regimes by numerically solving equation (3) and (4) with the initial wave function given by Eq. (5). In such, we focus on the interplay of nonlinearity, dispersion and dissipation affects the existence and properties of two-dark soliton in a polariton BEC.
We first briefly review some important features of a two-dark soliton in the coherent case, corresponding to the m R = 0 in Eq. (3). As a benchmark for later analysis, Equation (8) Then, we consider how the non-equilibrium nature of model affects the dynamics of two-dark-soliton solution when the dissipation parameters are turned on. In this end, we devise two scenarios: first, we choose a small initial velocity of v s and the two dark solitons will never penetrate but repel each other governed by the effective potential of V (x 0 ) in Eq. (9) when they are closer each other; then, when the initial velocity of v s is bigger than a critical value, the two dark solitons will overcome the effectively repulsive potential and penetrate. In the first scenario, we have chosen the initial soliton's velocity with v s = 0.15. As shown in Fig. 2 (c) and (d), the relative motion's minimum value is positive due to the reduction of their repulsive force between the solitons rooted into the interaction between atoms. Moreover, the numerical results (black solid curves) based on Eq. (8) find remarkable agreement with the analytically ones (blue dotted curves). Compared with Fig. 2 (b) without dissipation, the results with the introduction of dissipation in Fig. 2 (d) show that dark soltions are rebounded into farther positions, suggesting that the dissipation increases the repulsive effective potential. In the second scenario, the initial velocity of v s is chosen to be big enough to penetrate each other as shown in Fig. 2 (e) and (f). Note that our analytical results in Eq. (8) are valid under the condition that the relative distance of two solitons should be bigger than the width of soliton. Therefore our analytical results in Eq. (8) are supposed to be invalid when penetrating each other. In contrast, before and after collision of two dark solitons correspond to the relative distance of two solitons being bigger than the width of soliton, our analytical results in Eq. (8) are found to be consistent with the numerically ones as shown in Fig. 2
(e) and (f).
Conclusion.-In summary, we have investigated the dynamics of two dark solitons appearing in polariton BECs under nonresonant pumping. We have derived analytically the evolution equations for the solitons parameters. Within the framework of Lagrangian approach, our analytical results capture the essential physics about how the open-dissipative the effects affects the relative motion of two solitons at a finite time. We also solve the dissipative equation by the initial wave function of two dark solitons in a numerically exact fashion. The numerical results find remarkable agreement with the analytically ones. We also have investigated the collision of two solitons in polariton BECs under nonresonant pumping. By manipulating the initial velocity, the relative motion of the two solitons can repel or penetrate each other.
We thank Alexey Kavokin and Ying Hu for stimulating discussions. This work was supported by Zhejiang Provincial Natural Science Foundation of China (Grant No. LZ21A040001), the National Natural Science Foundation of China (No. 12074344) and the key projects of the Natural Science Foundation of China (Grant No. 11835011). | 2021-12-08T02:15:44.898Z | 2021-12-07T00:00:00.000 | {
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254021271 | pes2o/s2orc | v3-fos-license | The relationships between parents’ and children’s screen times on body mass index: a cross-sectional path analysis
Background Understanding factors contributing to an individual reducing screen time is essential for promoting a healthy weight. Parents’ behavior affects children by influencing their daily decision-making through modeling, rules or restrictions, social support, and co-participation. We examined how the direct and indirect effects of parents’ and children’s behaviors regarding screen time influenced body mass index (BMI) among Japanese elementary school children. Methods We included 283 Japanese children, one child per household, aged 6–12 years, who were randomly selected from resident registries of two cities. The questionnaires were completed by children and their mothers and fathers. Screen time and sociodemographic attributes, including sex, age, employment status, height, and weight, were assessed using a mail-based survey. Path analyses were conducted to determine associations among children’s, fathers’, and mothers’ variables. It was hypothesized that after controlling for household income and children’s sex and age, mothers’ and fathers’ screen time on weekdays and weekends would be related to children’s weekdays and weekend screen time, respectively. In addition, we hypothesized that children’s weekday and weekend screen time was related to children’s BMI. Results Both fathers’ and mothers’ weekday screen times were associated with children’s weekday and weekend screen times. BMI was affected by children’s weekday screen time (0.117). The path coefficients for the indirect effects of mothers’ and fathers’ screen time on children’s BMI through children’s weekday screen time were 0.016 from the fathers’ weekday screen time and 0.024 from the mothers’ weekday screen time (GFI = .980, AGFI = .953, RMSEA = .030, AIC = 93.030). Conclusions Both fathers’ and mothers’ weekday screen times indirectly affected children’s BMI through children’s weekday screen time among Japanese elementary school children. The strongest indirect effects could be seen by examining the paths of a mother’s weekday screen time through children’s screen time to BMI. Mothers who spend much time with their children are role models, and their behavior could affect the child’s behavior. The findings imply that intervention strategies to reduce screen time in children should also focus on modeling the mothers’ behavior.
Background
Obesity in children is a public concern worldwide and is associated with type 2 diabetes, hypertension, and an increased risk of obesity in adulthood [1,2]. For example, in Japanese school-aged children, 11.1% of boys and 8.8% Open Access *Correspondence: ishiikaori@waseda.jp 1 Faculty of Sport Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa 359-1192, Japan Full list of author information is available at the end of the article of girls aged 11 years were classified as obese in 2019 [3]. Compared to other developed countries, levels of obesity in Japanese school-aged children are low [4]; however, the percentage has grown in the last 10 years [3]. Especially in girls, elementary school-age students are more likely to be obese or overweight than junior high school or high school-age students [3]. Therefore, preventing obesity in children is vital for their future health.
Excessive sedentary behavior is associated with poor health and can result in increased adiposity, worse cardiometabolic health and fitness, impaired behavioral conduct/pro-social behavior, and reduced sleep duration [5]. For children, several current physical activity guidelines [6,7] recommend recreational screen time of no more than 2 h per day (i.e., watching television [T.V.], digital video discs, or videos, playing T.V. games, or using computers or the internet) and avoiding prolonged periods of sitting. Nevertheless, children spend too much time on their recreational screen time worldwide [8]. For instance, in the United States, 66% of children spend at least 2 h of screen time per day [9]. In Japan, approximately 60% of children have been found to exceed the 2 h per day mark of screen time [10].
Parents play an essential role in children's daily decision-making through modeling, rules or restrictions, social support, and co-participation [11,12]. Previous review studies have shown that parents' screen time is positively correlated with children's screen time [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27], and co-viewing with parents has been associated with increased screen time in children [28,29]. Moreover, the impact on children's screen time appears to be dependent on the sex of the guardian, as a previous study reported that mothers' screen-based behaviors showed a positive correlation with children's screen time [17,28,29]. However, few studies have considered gender differences in parental roles. Studies that have examined both the father's and mother's influence on children's sedentary behavior report that compared to the father's sedentary behavior, the mother's sedentary behavior influences the child's sedentary behavior more [28,29]. Xu et al. [30] concluded that reducing parents' screen time could decrease their child's screen time. Therefore, examining the impact of both fathers' and mothers' screen time on children is necessary.
In addition to the influence of the parents' gender, it has been reported that the influence of the parents' screen time on children's screen time varies between weekdays and weekends [19,27]. Jago et al. (2014) [27] concluded that associations observed between parent and child screen-viewing were different between weekdays and the weekend; they showed that on a weekday, children were 3.4 times more likely to exceed 2 h of screen viewing if their father watched T.V. for at least 2 h per day, while for a weekend day, children were 4.8 times more likely. There were similar associations for mothers; children were 3.7 times more likely to exceed 2 h of screen viewing if their mother watched T.V. for at least 2 h per day on a weekday, while children were 4.7 times more likely for a weekend. However, to our knowledge, only a few studies have examined the differentiation between weekdays and weekends [18,19,27].
The indirect effects and the strength of paternal and maternal screen time on children's screen time and body mass index (BMI) have not been examined. However, some studies have examined each of these variables directly, such as parents' screen time and children's screen time [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] or children's screen time and BMI [5]. Considering the impact of the behaviors of both father and mother on children in real life, parental behaviors may impact children's screen time and BMI, and suggestions for specific interventions to improve children's health might be possible through research. Thus, the present study examined how the direct and indirect effects of parents' and children's screen time behaviors influenced children's BMI among Japanese elementary school children.
Participants and data collection
The present cross-sectional study was conducted in a cohort of children living in Musashino City, Japan, in 2018 and Kokubunji City, Japan, in 2017. A total of 4800 potential residents aged 6-12 years, one child per household, were randomly selected from the residential registries of their respective cities. These selected children and their parents were the study participants. Musashino (population: 150,660 in October 2021) and Kokubunji (population: 130,636 in October 2021) are cities in Tokyo, Japan. Because both cities have approximately the same size of age population, the present study stratified them to account for this population ratio. Potential participants were stratified by sex (boys/ girls) and school grade (1st grade: 6-7 years, 2nd grade: 7-8 years, 3rd grade: 8-9 years, 4th grade: 9-10 years, 5th grade: 10-11 years, and 6th grade: 11-12 years). All mailings related to the mail-based survey were addressed to the children and their parents. First, invitation letters explaining the study were sent to all potential participants. A questionnaire and accelerometer were sent to those who responded to the invitation letter indicating they were willing to participate. To encourage a response, potential participants were told that a 1000-yen book voucher would be offered to those who returned the questionnaire and accelerometer. Non-respondents were sent one reminder about the responses to a questionnaire and accelerometer. A total of 1772 families (37.6% overall response rate; 881 responders from Musashino and 891 from Kokubunji) responded to the invitation. Then, self-administered questionnaires for children and their mothers and fathers were given, which included questions about sociodemographic variables, screen time, and children's height and weight, were mailed to those who responded that they would be willing to participate (620 individuals; 12.9% of all the invited respondents who mentioned they would be willing to participate, 310 people in each city). A total of 484 tryads completed the questionnaire and accelerometer measurements (78.1% overall response rate; 81.0% from Musashino, and 71.3% from Kokubunji). Data from 283 children and their parents who fully completed both questionnaires were included in the analysis (Fig. 1). Participation was voluntary, and confidentiality was ensured. A previous study [31] suggested that children younger than 10 years cannot report their activity patterns accurately or reliably. Alternatively, parental reports of physical activity among 6-year-olds have been shown to strongly correlate with heart rate measures during physical activity [32]. Therefore, parents of the children were asked to complete the questionnaire with their children. In addition, only mothers were asked to respond to the mothers' questionnaire and only fathers to the fathers' questionnaire.
Standard protocol approvals, registrations, and patient consent
All children, mothers, and fathers signed an informed consent form before answering the questionnaire. The Ethics Committee of Waseda University, Japan, approved the study before its commencement (2017-245). The present study was conducted in accordance with the principles of the 2013 Declaration of Helsinki.
Self-reported screen time
Domain-specific sedentary behaviors were assessed using a questionnaire. For children, sedentary behavior was divided into six domains [10] (1) reading or listening to music, (2) T.V. or video viewing, (3) T.V. game use, (4) internet or e-mail (computer or tablet) use outside of class, (5) doing homework or assignments, and (6) car travel for transport. Participants were asked how many days on average per week and how much time (hours and minutes) on average per day they engaged in these sedentary behaviors during weekdays and weekends in each domain. Then the weekly frequency was multiplied by the number of minutes per day. Each domain-specific sedentary behavior was examined separately, and we calculated the average total number of minutes for each school week (Monday-Friday) and weekends (days × minutes per day). Screen time was calculated using the total of domains (2), (3), and (4). The mothers and fathers were asked to report daily average sedentary time (hours and minutes) over the past 7 days, separately for workdays (weekdays for non-employed) and non-workdays (weekend for non-employed) across the following six domains: (1) being transported to and from a place by car; (2) using public transport; (3) at work; (4) watching television, videos, and DVDs; (5) using a computer, cell phone, and tablet P.C. outside of working hours; and (6) during leisure time (excluding watching television, videos, and DVDs) [33]. The total minutes of daily average screen time was calculated by summing (4) and (5) separately for workdays and non-workdays. Average daily values of sedentary time were calculated with weighting to account for the number of weekdays and weekend days.
Sociodemographic factors
Data on children's age and sex were collected from the residential registries. The children's current weights and heights were obtained from the questionnaire for children's responses. BMI was calculated from the height and weight data (BMI = weight/height [2]). Children's BMI percentiles were calculated using the metric system of the Centers for Disease Control and Prevention [34]. Children were classified according to the recommended BMI-for-age cutoffs [34]: < 5th percentile, underweight; 5-85th percentile, normal BMI; and ≥ 85th percentile, overweight or obese. Additionally, household income level per year (< 3, ≥3-< 5, ≥5-< 7, ≥7-< 10, or ≥ 10 million yen) and employment status (employed or not) were assessed from the parents' responses.
Statistical analyses
The data analysis involved assessing the replies from the 283 children, fathers, and mothers who had fully responded. Path analyses were conducted to determine the presence of any associations between the children's, fathers' , and mothers' variables. It was hypothesized that after controlling for household income and children's sex and age, mothers' and fathers' screen time during the weekdays and the weekend would be related to children's screen time during the weekdays and the weekend, respectively; it was also hypothesized that children's weekday and weekend screen time would be related to BMI. Path coefficients and correlations are reported as standardized estimates. The model was assessed using the goodness-of-fit statistic (GFI), adjusted goodness-offit statistic (AGFI), root mean square error of approximation (RMSEA), and Akaike information criterion (AIC).
GFI and AGFI indices were used to measure how well the model fit the data. Values of 0.90 or greater indicated a good model fit [35]. RMSEA is a measure of the descriptive measures of overall model fit. An RMSEA score value lower than 0.05 indicated a good fit [36]. A lower AIC value for a model indicated a better fit than the other models [37]. A model was considered to fit the data well when the following criteria were met: GFI > 0.90, AGFI > 0.90, RMSEA < 0.06, and a lower AIC value compared with competing models. Statistical significance was set at p < 0.05. The data were analyzed using path analyses estimated using IBM SPSS AMOS 27.0 J for Windows (IBM Corp., Armonk, N.Y., USA). Figure 2 shows the direct and indirect relationships between weekday and weekend mothers' and fathers' screen time, children's weekday and weekend screen time, and BMI. All path coefficients are standard partial regression coefficients. With the standard partial regression coefficients, the magnitude of each factor can be directly compared with the other factors in the model.
Direct and indirect effects of parents' and children's screen time behaviors on BMI
The present study identified no significant associations between (1) fathers' or mothers' weekday screen times and children's weekend screen times, or (2) between fathers' or mothers' weekend screen times and children's weekday screen times, or (3) between children's weekend screen times and BMI (GFI = 0.985, AGFI = 0.953, RMSEA = 0.029). Recalculating the model using modified indices reduced the AIC value from 96.351 to 93.030. Thus, the final model demonstrated an acceptable fit (GFI = 0.980, AGFI = 0.953, RMSEA = 0.030). Both fathers' and mothers' weekday screen times were seen to affect children's weekday screen times (from fathers' weekday screen times: 0.136, from mothers' weekday screen times: 0.203), and both fathers' and mothers' weekend screen times were seen to affect children's weekend screen times (from fathers' weekend screen times; 0.139, from mothers' weekend screen times; 0.271). BMI was affected by the children's weekday screen times (0.117). The path coefficient for the indirect effects of mothers' and fathers' screen times on BMI through children's weekday screen times was 0.016 from the fathers' weekday screen times and 0.024 from the mothers' weekday screen times.
Discussion
The present study indicated that both fathers' and mothers' weekday screen times indirectly affected children's BMI through children's weekday screen times among Japanese elementary school children.
There have been no consistent research findings on whether weekday or weekend screen times influence children's BMI. Only children's weekday (and not weekend) screen times were associated with BMI in the present study. A study of adolescents showed no significant correlation between weekday and weekend T.V. viewing times and BMI [38]; on the other hand, a study of preschool children showed that only children's weekend screen times were associated with BMI [18]. The small number of studies evaluating the effects on BMI that separated weekday and weekend screen times may have contributed to the lack of consistent results because whether it is a weekday or a weekend, a previous study [5] suggested that the total screen time has an impact on BMI. However, to reduce children's screen time, it would be helpful to consider what factors should be addressed on weekdays and weekends separately to adopt those concrete strategies. For example, a study of preschool children [18] reported that there was an association between screen time and BMI on weekends; however, the reason for no association between screen time and BMI on weekdays was that the total amount of time spent in front of a screen on weekdays was low. After all, the preschooler/ students went to kindergarten/preschool/school. In this study, the total screen time per week (9.4 hours on weekdays, 5.2 hours on weekends) was also significantly higher on weekdays than at weekends, which may be one of the reasons for this discrepancy.
Fathers' and mothers' weekday screen times influenced children's weekday screen time, which influenced BMI, and for both weekdays and weekends, the influence of a mother's screen time was stronger than that of a father's screen time. One previous study on the influences of parent screen time indicated that increased T.V. time of the child was associated with increased father's T.V. time (father odds ratio = 2.33 times, mother odds ratio = 2.24 times) than with mother's T.V. time [24]. The impact of fathers' T.V. time on children's T.V. time was stronger than that of mother's T.V. time in elementary school children (mean age 7.6 years), a similar cohort to the present study [24]. On the other hand, adolescents aged 12-13 years watching T.V. ≥ 2 hours per day were associated with mothers who watched T.V. ≥ 2 hours per day, but there was no association with fathers' T.V. time [28]. One of the reasons that these relationships were stronger for mothers than fathers in this study was that children in Japan spend more time with their mothers. A 2016 Ministry of Internal Affairs and Communications survey showed a [39]. Moreover, this trend seems to be associated with the employment rate of both parents in this study, as fathers were 33.2% more likely than mothers to be employed. Parents are role models for their children, and by shared time, their behavior could affect a child's behavior. Therefore, maternal modeling might have a stronger effect than paternal modeling on child behavior, as Japanese children spend much time with their mothers. Moreover, a previous study [19] that examined weekday and weekend screen times separately found no association between parents' and children's screen times; this study did not examine the fathers' and mothers' screen times separately. In contrast, another study [27] that examined weekday and weekday screen times found associations between children's weekday and weekend screen times and both fathers' and mothers' screen times on weekdays and weekends; this study examined the fathers' and mothers' screen times separately. In a study with at least one parent and a child aged 5-6 years, when parents exceeded 2 h of T.V. watching, children were 3.4 times and 4.8 times more likely to spend ≥2 hours T.V. watching during the weekdays and weekends, respectively, if their father exceeded the threshold; the odds were 3.7 for the weekdays and 4.7 for the weekends if their mothers exceeded the threshold [27]. This means that the influence of fathers and mothers may be different on weekdays and weekends. However, in the present study, although both fathers' and mothers' weekday screen times influenced children's weekday screen times and both the fathers' and mothers' weekend screen times influenced children's weekend screen times, the influence of the mothers' screen times was stronger than that of the fathers' screen times on both weekdays and weekends. Japanese children aged 10-14 years spend an average of 258 min with their mothers and 125 min with their fathers on weekdays, but on Sundays, they spend 411 min with their mothers and 272 min with their fathers; thus, although the children spend some time with their fathers, they spend 1.5 to 2 times longer time with their mothers [39]. Given this difference in Japan, the results of this study, which examined weekday and weekend screen times and paternal and maternal screen times separately, help develop intervention strategies to prevent obesity in Japanese children and reduce screen time.
Regarding the indirect effects on BMI, the strongest path was the influence of maternal weekday screen times on BMI via children's weekday screen times (0.024). On the other hand, fathers' indirect passes were 0.016. This may be partly due to the strong maternal commitment to weekday children's screen times, which suggests that mothers' influence should be considered when reducing screen time to improve BMI among Japanese children. Although the study did not examine fathers or mothers separately and was conducted in preschool-aged children, the indirect effect of parental screen time on children's BMI was found only on weekends [18]. In addition, the study that did not examine weekdays and weekends separately examined fathers' and mothers' physical activities separately and showed that the effect of mothers' physical activity on children's physical activity, which affects children's BMI, was significant, but the effect of fathers' physical activity was not [22]. There is a lack of research on modeling fathers' and mothers' behaviors on children's behaviors, and of children's behaviors on BMI; further research should be conducted in the future. For example, previous studies have reported efforts to reduce sedentary behavior in children and their parents through interventions targeting mothers and children [40]. There have been efforts to reduce parental sedentary behavior through parent education [41]. In the case of Japanese children, these interventions have not been reported. These efforts are expected to improve maternal literacy and reduce children's screen time, thereby improving their anthropometric indices.
Some limitations of this study should be considered. First, the study's cross-sectional nature limits the conclusions that can be drawn about the cause and effect of the observed relationships between parents' and children's screen times and anthropometric factors. Second, to estimate screen time, the study relied on self-reported measures with the potential for error owing to different interpretations of the questions. However, for children, the screen time scale has been used in a national survey of Japanese elementary school children [42], and the parent screen time scale confirmed its reliability and validity [33]. Third, the study respondents were slightly different from the general population. To estimate the representativeness of the participants' responses, the population percentage by age group in the present study was compared with data from population estimates [43]. The proportion of boys and girls aged 6-12 years was 45% (Kokubunji;44.6%, Musashino;45.1%) for boys and about 55% (Kokubunji;55.4%, Musashino;54.9%) for girls in this study, while the national data was about 51% for boys and 49% for girls. Therefore, the essential characteristics of respondents might have been biased. The findings in such a setting may not sufficiently apply to the general population. However, the present study randomly selected participants from a registry of each city's residential addresses, allowing an equal number of potential selects to be obtained from both sexes and each age group category between 1st grade and 6th grade. Although only sex and age distributions, the comparable variables investigated in this study, are compared, the present study population can be considered to have characteristics of the general population. Despite these limitations, few studies have been conducted on this topic in a randomly selected Japanese population, and the findings from the present study will contribute to a greater understanding of parental influences on children's screen time and anthropometric factors and may also help to develop new strategies and interventions to promote public health and well-being in Japan.
Conclusions
The present study indicates that both the fathers' and mothers' weekday screen times indirectly affected children's BMI through children's weekday screen times among Japanese elementary school children. The influence of the mothers' screen times was stronger than that of the fathers' screen times. The strongest indirect effects were seen by examining the paths of the mothers' weekday screen times via children's screen times to BMI. The total effect of the mothers' weekday screen times on BMI was 0.024. The present study's findings imply that intervention strategies to reduce screen time should also focus on mothers' modeling of children's health status. | 2022-11-28T15:02:32.290Z | 2022-11-28T00:00:00.000 | {
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3049398 | pes2o/s2orc | v3-fos-license | Classification of Older Adults Who Have Diabetes by Comorbid Conditions, United States, 2005–2006
Introduction Older adults who have diabetes vary widely in terms of comorbid conditions; these conditions help determine the risks and benefits of intensive glycemic control. Not all people benefit from intensive glycemic control. The objective of this study was to classify by comorbid conditions older American adults who have diabetes to identify those who are less likely to benefit from intensive glycemic control. Methods We used latent class analysis to identify subgroups of a nationally representative sample of community-dwelling older adults (aged 57–85 y) who have diabetes (n = 750). The subgroups were classified according to 14 comorbid conditions prevalent in the older population. Using the Akaike Information Criterion, the Bayesian Information Criterion (BIC), the sample-size adjusted BIC, and the χ2 goodness-of-fit statistic, we assessed model fit. Results We found 3 distinct subgroups. Class 1 (63% of the sample) had the lowest probabilities for most conditions. Class 2 (29% of the sample) had the highest probabilities of cancer, incontinence, and kidney disease. Class 3 (9% of the sample) had the highest probabilities (>90%) of congestive heart failure and myocardial infarction. Class 1 had only 0, 1, or 2 comorbid conditions, and both class 2 and class 3 had 6 or more comorbid conditions. The 5-year death rates for class 2 (17%) and class 3 (33%) were higher than the rate for class 1 (9%). Conclusion Older adults who have diabetes, cardiovascular disease, and 6 or more comorbid conditions may represent a subgroup of older adults who are less likely to benefit from intensive glycemic control.
Introduction
According to the American Diabetes Association (ADA), the glycemic target for most adults who have diabetes is a glycosylated hemoglobin (HbA1c) of less than 7.0% (1). Although the strategy of intensive glycemic control (defined as an HbA1c of <7%) may benefit many people who have diabetes, it may not benefit many older adults (2). Older adults who have diabetes are more heterogeneous than younger adults in terms of diabetes duration, functional ability, and comorbid conditions. The number and type of comorbid conditions may determine the risks and benefits of intensive glycemic control. The Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial heightened concerns about the harms of very intensive glycemic control (defined as an HbA1c <6.5%) among older adults (3). In light of these concerns, the current challenge is to identify older adults who are likely to benefit from intensive glycemic control.
Clinical markers may help identify these subgroups of older adults. For frail adults and adults who have a life expectancy less than 5 years, the American Geriatrics Society recommends less intensive glycemic control than that recommended by ADA (2). The 2010 ADA guidelines identify comorbid conditions, diabetes duration, hypoglycemia risk, and previous failures at intensive control as considerations for less intensive glycemic control (4). Despite these recommendations, no simple approach for identifying subgroups of older adults who would benefit from intensive glycemic control has been well accepted. For health systems, the identification of subgroups is especially difficult because many relevant markers (eg, hypoglycemia risk, mortality prediction, functional status) are not readily available from electronic medical records.
Classifying older adults who have diabetes according to comorbid conditions may be a practical strategy for identifying subgroups. Unlike methods requiring additional assessments (eg, functional status), data on comorbid conditions are readily available. Comorbid conditions are also associated with life expectancy and the degree to which intensive glycemic control provides benefits (5,6). The objective of this study was to classify by comorbid conditions the population of older adults in the United States who have diabetes in order to identify older adults who are less likely to benefit from intensive glycemic control.
Study design
We used latent class analysis (LCA) (7,8), the categorical analog to factor analysis, to identify subgroups by comorbid condition. We also compared their clinical characteristics and 5-year death rates. Institutional review boards at the Division of the Social Sciences at the University of Chicago and National Opinion Research Center approved data collection procedures. All respondents provided informed consent before participation in the study.
Study population
We used data from Wave 1 of the National Social Life, Health, and Aging Project (NSHAP), a longitudinal, populationbased study of health and social factors of older, community-dwelling Americans (9). Wave 1, conducted between July 2005 and March 2006 in English and Spanish, consisted of 3,005 interviews of adults aged 57 to 85; it oversampled men, black and Hispanic men and women, and men and women aged 75 to 84 at the time of screening. The sampling design also took into account urbanicity, defined as the probability-proportionate-to-size (PPS) selection of US Metropolitan Statistical Areas (MSAs) and non-MSA counties. NSHAP included an in-home interview, a biomeasure collection, and a self-administered postinterview questionnaire. Details on the NSHAP survey are available elsewhere (10,11). The Wave 1 survey had a weighted response rate of 75.5%, and the postinterview questionnaire had a weighted response rate of 84% (12).
We analyzed a subsample of 750 survey participants who self-reported diagnosed diabetes or who had undiagnosed diabetes. The following question was used to determine whether participants had diagnosed diabetes: "Has a medical doctor ever told you that you have any of the following conditions: [a list of conditions includes 'diabetes or high blood sugar']?" Participants who had an HbA1c of 6.5% or more and who did not self-report diabetes were considered to have undiagnosed diabetes (13). HbA1c was measured via dried blood spots during the biologic sample collection; details on the blood spot collection are available elsewhere (14). We chose not to determine diabetes diagnosis by examining data on prescribed medications because diabetes medications are also used to treat glucose intolerance.
The survey also collected self-reported data on age, sex, race/ethnicity, marital status, education, and number of visits with health care professionals in the previous year. We dichotomized number of health care visits to fewer than 4 visits in the previous year and 4 or more visits. Prescribed medications, including insulin, were cataloged during the in-home interviews (15). We measured self-rated physical health by using the single-item question, "Would you say your health is excellent, very good, good, fair, or poor?" Self-rated mental health was measured by a similar question. Responses to both questions were dichotomized (excellent/very good/good vs fair/poor). During the in-home interviews, participants also self-reported whether they had difficulty with each of the following activities of daily living for more than 3 months: walking 1 block, walking across a room, dressing, bathing/showering, eating, transferring (getting in/out of bed), and toileting. We collected data on 5-year death rates for the Wave 1 sample during Wave 2 of NSHAP during 2010 and 2011.
Latent class analysis
For LCA, we used data from all 750 survey participants. We included 14 chronic conditions that are prevalent in the older population (2,16): arthritis, cancer, congestive heart failure, dementia, depression, emphysema, falls (in the previous 12 months), hypertension, incontinence (urinary or fecal), kidney disease, myocardial infarction, obesity, stroke, and thyroid disease. All conditions were self-reported except depression and obesity, and all were assessed during the in-home interview except fall history. Depression was measured through the 11-item Iowa form of the Center for Epidemiological Studies Depression scale (CES-D). A score of 9 points or more on the CES-D was identified as depression (17). Obesity (body mass index [BMI] ≥30 kg/m2] was calculated from interviewer-measured height and weight (15). Fall history was assessed in the postinterview questionnaire.
We created separate variables for each unique combination of comorbid conditions among participants who had reported data for all conditions; 508 participants, 84% of the participants who returned the postinterview questionnaire, had complete data on all comorbid conditions. At most, 508 unique combinations of comorbid conditions were possible, one for each survey participant.
Statistical analyses
We fit latent class models successively, starting with a 1-class model and then adding another class for each successive model. Using the Akaike Information Criterion (AIC), the Bayesian Information Criterion (BIC), and the sample-size adjusted BIC (ABIC), we determined the optimal number of latent classes; lower values indicated better fit. Although the BIC is the most widely used criterion for assessing LCA model fit, multiple information criteria are often used in combination to select LCA class number (18). We also determined model fit by a χ2 goodness-of-fit P value greater than .05. We estimated models using full information maximum likelihood (FIML), which computes a case-wise likelihood function over the observed data by using all available information to estimate model parameters (19). FIML provides less biased and more efficient estimates than pair-wise deletion, case-wise deletion, or similar responsepattern imputation (20). We compared model fit between 1-, 2-, 3-, and 4-class models. We used Mplus version 6 (Muthén & Muthén, Los Angeles, California) to conduct the analyses.
We used χ2 analysis and 1-way analysis of variance to describe class differences in sociodemographic characteristics, self-rated health, health care use, clinical characteristics, including the prevalence of multiple comorbid conditions, and 5-year death rates. We also compared classes by frequency and combinations of comorbid conditions. We used STATA version SE10.1 (StataCorp LP, College Station, Texas) to conduct these analyses. All analyses were weighted by using population weights that adjusted for the intentional oversampling of black and Hispanic participants and incorporated a nonresponse adjustment based on age and urbanicity. We adjusted standard errors for sample stratification (sampling strata independently) and clustering (sampling individuals within each of 100 primary sampling units).
Model fit
The AIC, BIC, and ABIC decreased as the number of classes increased from 1-to 4-class models ( Table 1). The AIC, BIC, and ABIC were all lowest with the 4-class model, but the AIC and ABIC only marginally decreased between the 3and 4-class models, and the 4th class had only 7 members. The P value for the χ2 goodness-of-fit statistic was greater than .99 for all 4 models. Thus, we selected a 3-class model to distinguish between population subgroups.
Class characteristics
The estimated probability of having obesity, hypertension, or arthritis was at least 45% in all 3 classes (Table 2). Most participants (n = 470, 63%) were in class 1, which was characterized by the lowest probability for nearly all conditions. The estimated probability of having dementia, congestive heart failure, emphysema, kidney disease, or stroke was less than 5% in class 1. Approximately 29% of participants (n = 215) were in class 2. Class 2 had the highest estimated probabilities of cancer, hypertension, incontinence, kidney disease, and obesity. Class 3 (n = 65, 9%) had the highest estimated probabilities of arthritis, congestive heart failure, depression, emphysema, falls, myocardial infarction, stroke, and thyroid disease. The estimated probability of congestive heart failure or myocardial infarction was more than 90% for class 3. Class 2 had the lowest average HbA1c (Class 1, 7.24%; class 2, 6.86%; class 3, 7.28%) ( Table 3). A higher percentage in class 2 and class 3 reported 4 or more visits with health care professionals in the previous year than in class 1. More than half of class 2 (59%) and class 3 (78%) reported fair or poor physical health, compared with 26% of class 1. In addition, class 2 and class 3 had more difficulty than class 1 with all activities of daily living. For example, 54% of class 2 and 66% of class 3 reported difficulty walking 1 block, compared with 24% of class 1. The 5-year death rate of class 3 (33%) was almost twice the rate of class 2 (17%) and 3 times the rate of class 1 (9%). We found 266 combinations of comorbid conditions. Class 1 had 116 combinations, class 2 had 118 combinations, and class 3 had 32 combinations; we found no overlap of combinations between classes. The most frequent combinations of conditions were arthritis, hypertension, incontinence, and obesity (n = 18) and arthritis, hypertension, and obesity (n = 15).
Discussion
In this nationally representative sample of community-dwelling older adults who have diabetes, we found 3 classes of people, based on their comorbid conditions: people in class 1 (63% of the sample) had 2 or fewer comorbid conditions and were relatively healthy; people in class 2 and class 3 were sicker; they had 6 or more comorbid conditions, a higher 5-year death rate, more health care visits, and higher rates of insulin use, functional disability, and fair or poor selfrated health. Classes were not distinguished by any single comorbid condition. However, in class 3, the estimated probability of myocardial infarction was 93% and congestive heart failure, 100%; one-third of respondents in class 3 died within 5 years of participating in the first wave of the study.
The ADA (4) recommends that clinicians use information on comorbid conditions to identify older adults who are less likely to benefit from intensive glycemic control. However, because comorbid conditions co-occurred in many different patterns across all classes, the task of identifying patients by comorbid conditions in clinical practice would be challenging. Instead, we found a potentially simpler strategy. Using 2 categories based on the number of comorbid conditions (high [≥6] and low [≤2], one could assign nearly half of the participants in our study (n = 232, 46%) to the correct class. Thus, older adults who have diabetes and 6 or more comorbid conditions would be unlikely to benefit from intensive glycemic control.
The presence of cardiovascular disease may raise concerns about the effects of intensive glycemic control. In the ACCORD trial, the presence of cardiovascular disease appeared to decrease the benefits of intensive glycemic control (3). In the Veterans Affairs Diabetes Trial, the presence of coronary calcification was associated with higher rates of cardiovascular events during very intensive glycemic control (21). These recent findings would have direct implications for our class 3 participants, for whom the estimated probability of cardiovascular disease was more than 90%. Although most participants in class 3 had cardiovascular disease, participants in class 1 and class 2 also had cardiovascular disease. Thus, decisions about intensive glycemic control should take into account both counts of comorbid disease and the presence of sentinel conditions, such as cardiovascular disease.
This research has several strengths and limitations. It demonstrates that distinct subgroups of older adults who have diabetes share patterns of comorbid conditions. It partially simplifies the complex heterogeneity of comorbid conditions among older adults who have diabetes. Our classification system, based on 2 categories, could guide future geriatric diabetes treatment recommendations. One limitation to this work is that we were unable to account for the likely important effects of diabetes duration. Differences in duration have been proposed to explain the differences between the United Kingdom Prospective Diabetes Study (22) and more recent trials. Although too late for our study, NSHAP now collects self-reported data on diabetes duration. One limitation to using self-reported data on diabetes duration, however, is the delay between diabetes onset and diabetes diagnosis; previous trials have defined the onset of diabetes by clinical diagnosis (1,3,21). Future longitudinal data would allow examination of the development of new comorbid conditions in subgroups of older adults who have diabetes. Finally, because information on fall history was collected through the postinterview questionnaire and not during the in-home interview, this information may have been systematically underreported, and the lack of these data reduced the number of people in our analysis.
Classifying the older US population who have diabetes on the basis of comorbid conditions produces clinically distinctive subgroups; however, these subgroups overlap in the predicted probability of comorbid conditions. Relying on classes based on comorbid conditions alone may not provide a definitive classification system for this population. However, using information on cardiovascular disease history and counts of comorbid conditions could provide strategies to clinicians and policy makers for distinguishing clinically important subgroups. Future research is needed to evaluate alternative strategies for identifying the subgroup of older adults who have diabetes in whom the risks of intensive glycemic control outweigh its benefits. | 2017-06-19T08:31:31.203Z | 2012-05-17T00:00:00.000 | {
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3851447 | pes2o/s2orc | v3-fos-license | Antidyslipidemic potential of a novel farnesoid X receptor antagonist in a hamster model of dyslipidemia: Comparative studies of other nonstatin agents
Abstract We attempted to clarify the therapeutic capability of antagonists of the farnesoid X receptor (FXR), a nuclear receptor that regulates lipid and bile acid metabolism. Herein, we report the antidyslipidemic effects of a novel synthesized FXR antagonist, compound‐T1, utilizing a dyslipidemic hamster model. Compound‐T1 selectively inhibited chenodeoxycholic acid‐induced FXR activation (IC 50, 2.1 nmol·L−1). A hamster model of diet‐induced hyperlipidemia was prepared to investigate the antidyslipidemic effects of compound‐T1 through comparative studies of the nonstatin lipid‐modulating agents ezetimibe, cholestyramine, and torcetrapib. In the hamster model, compound‐T1 (6 mg·kg−1·day−1, p.o.) increased the level of plasma high‐density lipoprotein (HDL)‐cholesterol (+22.2%) and decreased the levels of plasma non‐HDL‐cholesterol (−43.6%) and triglycerides (−31.1%). Compound‐T1 also increased hepatic cholesterol 7α‐hydroxylase expression and fecal bile acid excretion, and decreased hepatic cholesterol content. Moreover, the hamster model could reflect clinical results of other nonstatin agents. Torcetrapib especially increased large HDL particles compared with compound‐T1. Additionally, in the human hepatoma Huh‐7 cells, compound‐T1 enhanced apolipoprotein A‐I secretion at a concentration close to its IC 50 value for FXR. Our results indicated the usefulness of the hamster model in evaluating FXR antagonists and nonstatin agents. Notably, compound‐T1 exhibited beneficial effects on both blood non‐HDL‐cholesterol and HDL‐cholesterol, which are thought to involve enhancement of cholesterol catabolism and apolipoprotein A‐I production. These findings aid the understanding of the antidyslipidemic potential of FXR antagonists with a unique lipid and bile acid modulation.
| INTRODUCTION
High blood cholesterol is a risk factor for cardiovascular events. 1,2 3-Hydroxymethyl 3-glutaryl-CoA reductase inhibitors, also known as statins, markedly reduce both blood cholesterol and cardiovascular risk in hyperlipidemia patients. [3][4][5] However, for patients who cannot achieve their cholesterol-lowering targets with statins, owing to insufficient efficacy or adverse effects, nonstatin agents are also effective as an alternate or add-on therapy to statins. 6 One such nonstatin agent is cholestyramine, a bile acid sequestrant, which forms a complex with bile acids in the intestine and is then eliminated into feces. Through the augmentation of bile acid synthesis and cholesterol catabolism, cholestyramine demonstrated a significant reduction in blood cholesterol. 7,8 A recent meta-analysis and large-scale epidemiologic analysis suggested that the cholesterol-lowering effects of bile acid sequestrants, including cholestyramine, may contribute to the prevention of cardiovascular diseases. 9 Another nonstatin medication is the cholesterol absorption inhibitor ezetimibe, which targets intestinal uptake of dietary and biliary cholesterols; good efficacy and tolerability of combination therapy with statin and ezetimibe have been reported. 10 Another approach aimed at the reduction in cardiovascular risk is the increase in blood high-density lipoprotein (HDL)-cholesterol levels 11 by the inhibition of cholesteryl ester-transfer protein (CETP), which mediates transfer of cholesteryl ester from HDLs to atherogenic lipoproteins. Clinical trials involving torcetrapib, the first CETP inhibitor, were terminated early owing to off-target toxicity, 12,13 and despite remarkable increases in HDL-cholesterol, torcetrapib did not reduce the progression of atherosclerosis. 14,15 The definitive clinical efficacies of CETP inhibition are under investigation in the ongoing REALIZE study of anacetrapib, a more recent CETP inhibitor. 16 As a possible concept for another novel nonstatin agent, we focused on the inhibition of farnesoid X receptor (FXR), a member of the nuclear receptor superfamily. FXR is expressed at high levels in tissues involved in bile acid metabolism such as the liver, intestine, and kidney. 17,18 In the liver, FXR indirectly suppressed expression of cholesterol 7a-hydroxylase (CYP7A1), a rate-limiting enzyme in the cholesterol catabolic pathway, 19 and also directly downregulated the expression of apolipoprotein A-I, a major protein constituent of HDL. 20 We therefore hypothesized that repression of FXR would improve dyslipidemia via both non-HDL-cholesterol reduction and HDL-cholesterol elevation, and we successfully confirmed that a synthesized FXR antagonist, compound-T3, displayed these blood changes in cynomolgus monkeys. 21,22 In this study, to clarify the therapeutic advantage of an FXR antagonist over the nonstatin agents ezetimibe, cholestyramine, and torcetrapib, we investigated Figure 1) and conducted comparative studies in a high-fat diet-induced dyslipidemic hamster model.
| Nuclear receptor panel assays
The agonistic and antagonistic activities of human nuclear receptors, FXR and PPARs PPARa, PPARd, and PPARc were measured as previously described. 23,24 The agonistic and antagonistic activities for the human retinoid X receptor-a (RXRa) and the human liver X receptors (LXRs) LXRa and LXRb were also measured. Briefly, the reporter construct for the RXRa reporter gene assay, pGL3-DR1 9 4-tk-luc, was created by the insertion of four copies of direct repeat 1 element (AGGTCA-N-AGGTCA) upstream of the herpes virus thymidine kinase promoter and the luciferase reporter gene. To construct the LXRa and LXRb reporter gene assays, pGL3-DR4 9 4-tk-luc was generated by the insertion of four copies of direct repeat 4 (AGGTCA-NNNN-AGGTCA) at the same position as pGL3-DR1 9 4tk-luc.
| Multiple-dose study
Hamsters were fed with a high-fat diet (a chow diet containing 10.3% nonsalt butter, Oriental Yeast Co., Ltd) and housed individually in cages after the age of 6 weeks. After high-fat diet feeding for 1 week, drug treatments were commenced at the age of 7 weeks.
Food consumption was determined for each animal every day. Compound-T1, ezetimibe, and torcetrapib were suspended in 0.5% immediately into storage at À30°C for lipid level measurements and at À80°C for gene expression measurements.
| Measurement of plasma C4 levels
Plasma C4 levels were measured following the method described in our previous report. 23 Briefly, 25 lL plasma was mixed vigorously with 10 lL of 7b-hydroxy-4-cholesten-3-one (50 lgÁmL À1 in dimethylsulfoxide; Calbiochem, USA), as an internal standard, and 500 lL acetonitrile, and then the mixture was sonicated for 5 minutes. After centrifugation, 100 lL supernatant was processed using a HPLC system (ClassVP; Shimadzu, Japan) fitted with a Nova-Pak C18 steel column (Waters Corporation, USA), and analyzed with 95% acetonitrile (flow rate: 1 mLÁmin À1 ) as the mobile phase. The absorbance of C4 was detected at 241 nm; absorbance areas of C4 and the internal standard were calculated, and the concentrations of C4 were quantified against an internal standard.
| Measurement of hepatic lipid levels
The frozen liver samples (approximately 1.0 g) were homogenized by the addition of 3.35% sodium sulfate solution (9 mL) and shaken for 10 minutes after the addition of a 3:2 solution of hexane-isopropanol (3:2) solution. Subsequently, 100 lL of the solution was evaporated to dryness and solidified at 50°C in a nitrogen atmosphere to form a dried residue. The residue was dissolved in a 100 lL of diox- Table S1). Expectedly, these three agents Table S2). Torcetrapib significantly elevated HDLcholesterol level but did not lower non-HDL-cholesterol and TG levels, which was in contrast to compound-T1. Although no changes in body weight were observed in all groups, a significant reduction in food intake was observed in the 10 mgÁkg À1 Áday À1 of compound-T1-treated group (À22.5%, data not shown).
To perform a detailed comparison between the HDL-cholesterolelevating effects of compound-T1 and torcetrapib, we conducted lipoprotein profiling by HPLC gel filtration system ( Figure 5). The selectivity of compound-T1 for human nuclear receptors was measured as described in the Methods section. change in the peak value of particle size. At doses of 3 mgÁkg À1 Áday À1 and 10 mgÁkg À1 Áday À1 , compound-T1 also increased HDL levels with no change in the peak value of particle size, but an increase larger HDL particles (fraction number 16) was observed, especially at the higher treatment concentration. In contrast, torcetrapib produced a more marked increase in HDLs with a larger particle diameter; in particular, a dose of 100 mgÁkg À1 Áday À1 of torcetrapib resulted in an increase in much larger particles (fraction number [13][14][15], which were hardly observed in the control and compound-T1-treated groups. Torcetrapib did not produce any changes in the VLDL and LDL fractions.
| Comparative studies on fecal and hepatic lipid profiles in a dyslipidemic hamster model
The changes in fecal lipid content after treatment with compound-T1, ezetimibe, and cholestyramine are summarized in Figure 6A.
Both compound-T1 and cholestyramine significantly increased fecal total bile acid content, while ezetimibe increased both fecal total cholesterol and TG content. No other significant changes were observed in all groups.
Next, the changes in fecal lipid content were compared between compound-T1 and torcetrapib, and are summarized in Figure 6B.
Compound-T1 dose-dependently increased fecal total bile acid content, while torcetrapib resulted in a significant decrease. A significant reduction in fecal total cholesterol was observed in both groups receiving the highest dosage of compound-T1 (10 mgÁkg À1 Áday À1 ) and torcetrapib (100 mgÁkg À1 Áday À1 ). There was no significant change in fecal TG in all groups.
The effects of compound-T1, ezetimibe, and cholestyramine on hepatic lipid content are summarized in Figure 7. Compound-T1 dose-dependently lowered hepatic total cholesterol and cholesteryl ester. Compound-T1 also decreased hepatic free cholesterol at a dose of 10 mgÁkg À1 Áday À1 . Torcetrapib did not show any significant change in hepatic total cholesterol, cholesteryl ester, and free cholesterol content. In addition, hepatic TG content was decreased in compound-T1-treated group, but increased in the 100 mgÁkg À1 Áday À1 torcetrapib-treatment group.
To confirm the FXR antagonistic activities of compound-T1 after multiple doses, we measured the hepatic expression levels of CYP7A1. Hepatic CYP7A1 mRNA levels were dose-dependently increased in the compound-T1-treated groups and in the cholestyramine-treated group. There were no significant changes in the ezetimibe-treated groups (Figure 8).
| Effects of compound-T1 on apolipoprotein A-I production in Huh-7 cells
To elucidate the mechanism of the HDL-cholesterol-elevating effects of compound-T1, we investigated its effect on apolipoprotein A-I production using the human hepatoma Huh-7 cell line under physiological CDCA conditions. Apolipoprotein A-I secretion was significantly reduced by the addition of 30 lmolÁL À1 CDCA, and this reduction was dose-dependently rescued by compound-T1 with an ED 50 value of 1.8 nmolÁL À1 (Figure 9).
| DISCUSSION
We have previously confirmed that a synthesized FXR antagonist, compound-T3, reduced plasma non-HDL-cholesterol levels and also elevated HDL-cholesterol levels in a primate model. 21,22 However, the development of a rodent model with similar disease conditions to human hyperlipidemia would be still useful for the further comprehension of this new type of agent. However, the rodent animals such as mice, rats, and guinea pigs have different plasma lipid and lipoprotein profiles from humans. 23,25 Actually, we found that another FXR antagonist, compound-T0, exacerbated dyslipidemia in mice due to enhancement of intestinal lipid absorption via acceleration of bile acid excretion. 26 We think that the difference of the effects of FXR antagonist between primate and mouse models would be due to the difference in regulation of key players relating to lipid and bile acids metabolisms, in particular, the hepatic LDL receptor. 27 In addition, CETP is also a key protein involved in plasma cholesterol transport that transfers cholesteryl ester from HDL to LDL and VLDL. 28 Golden Syrian hamsters are known to develop human-like hyperlipidemia following the feeding of a high-fat diet, [29][30][31] and also show both plasma CETP activity 28 and regulation of hepatic LDL receptor. 32 Therefore, we considered that a hyperlipidemic hamster model would be suitable for the purpose described above. To determine the appropriate diet for hamsters, we referenced diet information in nonclinical and clinical studies of orlistat, an antiobesity drug. 33,34 After the hamsters were fed this butter-rich diet for a week, both plasma levels of cholesterol and triglyceride increased to approximately two and three times the normal level respectively F I G U R E 5 Effects of compound-T1 and torcetrapib on plasma lipoprotein distribution in high-fat diet-fed hamsters. Pooled plasma (n = 6 per group) was collected after repeated drug administrations and then was fractionated by gel filtration HPLC. The measurement procedures are described in the Methods section SHINOZAWA ET AL. (Table S3). Using this hamster model, herein, we sought to clarify the favorable pharmacological profile of a novel FXR antagonist compound-T1, a potential antidyslipidemic agent, through comparative studies with other nonstatin agents ezetimibe, cholestyramine, and torcetrapib.
The first question in our study was to determine whether the clinical results of other nonstatin agents were consistent in our hamster model. Firstly, ezetimibe, a cholesterol absorption inhibitor, exhibited clinically relevant changes with regard to the plasma non-HDL-cholesterol reduction and fecal cholesterol excretion. 35,36 Although a significant increase in fecal TG was also found in ezetimibe-treated hamsters, as reported in a previous study using an obese rodent model, 37 the dramatic effect of ezetimibe on TG excretion has not been reported in a clinical setting. However, an inhibitory effect on the intestinal production of chylomicrons, which consist largely of TG, was reported in patients with hyperlipidemia. 38 Therefore, we assumed that the increased fecal TG in ezetimibe-treated hamsters might result from its intervention in chylomicron TG F I G U R E 9 Effect of compound-T1 on apolipoprotein A-I secretion in CDCA-treated Huh-7 cells. Apolipoprotein A-I concentration in cell supernatant was measured with a sandwich ELISA method, as described in the Methods section. Each value represents the mean AE SEM (n = 3). Statistical analysis was carried out using Student's t-test (**P ≤ .01 vs control) or one-tailed Williams' test ( † P ≤ .025 vs CDCA(+)) observations as described above. A high dosage requirement can sometimes affect patient drug compliance. 46 In this respect, compound-T1 would be therefore superior to cholestyramine. Additionally, in the present study, compound-T1 did not result in a large change in HDL particle size, but torcetrapib did. Future studies are needed to clarify this, but it would be important to investigate whether increased HDL particles can promote the reverse cholesterol transport system to cause cholesterol efflux from atherosclerotic lesions to the liver and feces; in particular, we recommend a focus on apolipoprotein A-I, which has been reported to be a key factor in the effective promotion of beneficial HDL metabolism. 47 Indeed, intravenous infusion of a variant of apolipoprotein A-I-phospholipid complexes resulted in the regression of atherosclerosis in patients with acute coronary syndromes. 48
DISCLOSURE
The authors are employees of Takeda Pharmaceutical Co., Ltd (Osaka, Japan) at which Compound-T1 was synthesized. | 2018-04-03T02:37:30.072Z | 2018-03-08T00:00:00.000 | {
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255692766 | pes2o/s2orc | v3-fos-license | The opioid crisis as it pertains to spine surgery
reduces
We read a recent study from Rezaii et al. with great interest: "Lumbar spine surgery reduces postoperative opioid use in the veteran population" (1). We commend the authors on their work within the veteran population who concluded that veterans, despite being a high-risk population with a higher burden of mental health comorbidities, have a postoperative opioid dependence that mirrors the general population following lumbar spine surgery. In addition, results demonstrated that preoperative opioid exposure increased the likelihood of opioid use one year after surgery.
Furthermore, their results stressed the importance of proper preoperative opioid counseling and weaning strategies postoperatively. A study conducted at our institution examined opioid use after elective spine surgery in the general population (2). Our results echo Rezaii et al. with regards to the impact of preoperative opioid exposure on the degree of opioid use postoperatively. Additionally, our results demonstrated that the modern elective spine surgery patient's opioid utilization after surgery is less than routinely prescribed. Thus, our intent in this editorial is to briefly review the history of the opioid epidemic, its relation to orthopaedic surgery, and to provide remarks on mitigating strategies with regards to spine surgery.
The opioid epidemic history & background
In 2017, the U.S. Department of Health and Human Services (HHS) declared a public health emergency to address the national opioid crisis after more than 40,000 deaths were attributed to opioid overdoses, which alarmingly exceeded all preceding years. In 2019, over 70,000 people died from all drug overdoses; 14,000 of those deaths involved prescription opioids, averaging 38 people dying each day from overdoses involving prescription opioids. More recent statistics show that in 2020: 10.1 million people misused prescription opioids and there were 1.6 million sufferers of opioid use disorder in the United States alone. The total economic burden of prescription opioid misuse in the United States is an estimated $78.5 billion per year according to the Center for Disease Control and Prevention (3)(4)(5)(6).
The opioid epidemic dates back to the 1990's when physicians began increasing their opioid prescribing practices. This change in practice has been attributed in part by pharmaceutical companies misleading prescribers that the risk of addiction to some opioid pain relievers was extremely small (7). When it became clear that these medications were highly addictive, the damage was already done, as many of the large quantity of opioids prescribed became misused, abused, or diverted. At the same time, the American Pain Society declared pain as the "fifth vital sign" supporting the evaluation and treatment of pain, which was deemed as essential as monitoring temperature, blood pressure, respiratory rate, and heart rate (8,9). In 2001 this
Editorial
The opioid crisis as it pertains to spine surgery idea was supported by the Joint Commission as part of their Pain Management Standards, where pain control was put under the microscope.
The opioid epidemic continues
Opioid prescriptions given to control pain after surgery are often the first time a patient is exposed to opioids and is a well-documented risk of chronic opioid dependence in the opioid naïve. However, the majority (70%) of prescription opioid users receive them through diversion, often from legitimate prescriptions of friends and family (8). Many surgeons prescribe opioids to control postoperative pain, with orthopedic surgeons rated as the third highest prescribers of opioids (10)(11)(12). Miscalculating the magnitude of postoperative pain can lead to over-prescribing opioids after surgery, thereby inadvertently contributing to the quantity of unused pills available for diversion (2).
Spine surgeons are faced with four unique challenges in managing their patients' pain. First, spine surgery is associated with substantial postoperative opioid requirements and consumption, demonstrated in several recent studies evaluating opioid use after a variety of elective orthopedic and non-orthopedic surgeries (8,10,13,14). Second, according to data extracted from the Global Burden of Disease, Injuries, and Risk Factors Study between 1990-2017, low back pain was the leading cause of disability globally and continues to remain among the top 5 causes of disability worldwide (15,16). Third, many patients are prescribed opioids prior to seeking treatment from a spine surgeon, therefore they are no longer opioid naïve and have greater risk of opioid misuse, abuse, and developing opioid dependence. Finally, the aging population with a longer life expectancy coupled with more minimally invasive spine surgery options has led to a greater number of spine surgeries being performed each year, estimated at nearly 1 million cases in the United States annually (17).
Combating the opioid epidemic today
Despite these hurdles, spine surgery continues to evolve in significant and positive ways. The advent of minimally invasive spine surgery has brought robotic-guided, navigation-guided, augmented reality-assisted, and endoscopic spine surgery options which have been shown to reduce tissue disruption and operative times, giving the potential for a reduction of hospital length of stay and a faster recovery overall (18)(19)(20).
In addition to these innovative surgical techniques, implementation of enhanced surgical recovery (ESR) protocols has modernized the way spine surgery patients prepare for, undergo, and recover from surgery (21,22). Combining preoperative education on pain management and weaning after surgery, optimizing intraoperative nonopioid pain-relieving tactics, and a multi-modal approach to pain management postoperatively, adequate pain control can be achieved while opioid use is minimized after surgery.
Furthermore, we are able to better predict opioid utilization patterns by identifying certain patient characteristics, such as (I) preoperative opioid exposure, (II) surgery type, (III) age, (IV) BMI, (V) depression/anxiety diagnoses, (VI) length of hospital stay, and (VII) pain scores at hospital discharge. With this information, tailoring prescribing practices to the individual's needs is possible.
It is important to recognize that contributors to the opioid crisis are multifactorial, with social and economic determinants of health playing a major role in its trajectory (23). We acknowledge that supply and overdoses attributed to illicit fentanyl and heroin may be the lead driving force in today's version of the crisis, yet it is important to focus on all facets of the crisis, however small they may be.
Conclusions
The Opioid Epidemic presents a serious challenge to orthopaedics and the US healthcare system overall. Adjustments in prescribing practices and patient education are paramount in combating this facet of the epidemic. The advent of innovative technologies, minimally invasive surgical techniques, and ESR protocols present promising opportunities in addressing this crisis as it relates to spine surgery. Additional high-quality studies continue to be necessary to provide further information on this timely topic. | 2023-01-12T17:40:45.305Z | 2022-01-01T00:00:00.000 | {
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256679530 | pes2o/s2orc | v3-fos-license | Structural Study of the Precambrian Basement of Kaele (Far North Cameroon): Contribution of Remote Sensing (Application of Landsat 8 OLI/TIRS Images) and Field Data
The Kaele is located in the northern domain of the Pan-African fold belt of Central Africa in Cameroon, north of Mayo- Kébbi domain. The structural study carried out in this area is based on Landsat 8 OLI/ TIRS image processing techniques and traditional geological prospection methods. The objective is to evaluate the contribution of landsat 8 OLI/TIRS image processing and field data in the structural mapping of the Kaélé region. The application of the 7×7 directional filters of the Sobel type in the N-S, E-W, NE-SW and NW-SE directions, to the ETM+3 channels gives better results. The rose diagram of the lineament map (obtained from image processing) shows a major E-W direction (N100E) and a secondary direction N-S (N10E). The studied area is affected by a polyphase deformation D1 to D4. The first phase of deformation (D1) is tangential and has set up the sub-horizontal dipping S1 schistosity which bears a composite L1 lineation (stretching, mineral). The D2 phase is constrictional and responsible for the establishment of the subvertical S2 axial plane foliation of the P2 folds, carrying the composite L2 lineation (stretching, mineral) and the B2 boudinage. The dextral-moving C2 shear cuts the S1 foliation, the P2 folds are asymmetrical isopach and anisopach. The third phase D3 is a constriction and set up the S3 schistosity which appears in syn -tectonic granite and highlight the C3 shear planes. The C3 shear is dextral and the P3 folds are isopach, anisopach, intrafolial and sheath fold. The last phase of deformation D4 is brittle and has set up faults and joints. There exist similarities between orientation of lineament from image processing and direction of D2, D3 and D4 deformational phases structures.
Introduction
The Central African fold belt (CAFB) (Figure 1a) is an elongated orogenic mega-belt trending E-W, more than 5000 km long and 300 km wide; it cover over Nigeria, Cameroon, Chad, Central African Republic [1,2,3] and extends to the NE of Brazil in the province of Borborema by the Brazilian chain [4][5][6][7][8][9]. The CAFB is subdivided in Cameroon into three domains [10]: the Southern domain, the Central domain and the Northern domain. The Northern domain includes a NW sub-domain and a Mayo-Kébbi sub-domain [11] ( Figure 1b). The study area is located in the Mayo-Kébbi sub-domain. [11,13,14]) showing the major lithological units and the study area: (1) Post-pan-African sediments; (2) Post to late tectonic Pan-African Granitoids; (3) Syntectonic granite; (4) Mayo-Kébbi Batholith; (5) medium to high grade Gneiss of the Northern domain; (6) Mafic complex of the Mayo-Kébbi domain (metadiorite and gabbro-diorite); (7) Neoproterozoic volcano -sedimentaries Sequences of the Poli-Léré group; (8) Adamaoua-Yade Paleoproteozoic domain; (9) thrust; (10) FTB= Tcholliré Banyō fault; FGG=Godé-Gormaya fault; (11) Border limit Remote sensing and image processing have imposed themselves in recent years in geological mapping, due to the scarcity of outcrops, their discontinuity and the inaccessibility of their limits. The diversity of satellite sensors and their technical characteristics (spatial, spectral and temporal resolutions) provide additional information that can be combined with data from other sources [12], particularly field data. This study aims to apply remote sensing and field data in the structural study of the Kaélé region.
Geological Setting
The Mayo -Kébbi domain which covers Cameroon and Chad is an NE-SW elongated unit. It is located between the Adamawa-Yade domains and NW Cameroon domain (see Figure 1b). This domain is limited to the SE by the Tcholliré-Banyo fault in Cameroon and its extension, Massenia-Ounianga gravimetry anomaly, in Chad [11,15]. It is separated to the NW from the NW Cameroon domain by the volcano-sedimentary series of Zalbi, considered as a NE extension of the Poli group [8,11,16]. The Mayo-Kébbi domain differs from the Adamawa-Yade domain and NW Cameroon domain by its juvenile Neoproterozoic crust [11,17,18]. This domain corresponds to a magmatic arc zone which was formed between 800 Ma and 550 Ma by successive collision with the Central Cameroon and NW domains respectively [11,17,18]. It consists of: (1) the greenstone belts formed by the volcano-sedimentary series and the mafic to intermediate complex, (2) the Mayo-Kébbi batholith, and (3) the post-tectonic intrusions [11,17,18,19,20,21]. Study of the deformation highlights three tectonics phases D1, D2 and D3. There is a predominance of the NNE-SSW elongated direction. The first two are syn-schistose and the last is essentially strike-slip [19].
Materials
Envi 5.2, Geomatica 2015 and Qgis 3.18 softwares were used for multi-spectral image processing and analysis. Qgis 3.18 software was also used for georeferencing, digitizing and adding maps to the database. StereoWin1.2 and CorelDraw12.0 software were used for field data processing.
Data Used
The cartographic database used for this study includes Landsat 8 image, the scene acquired between April 27, 2021 and May 01, 2021 under favorable climatic conditions to the visualization of geological elements on the ground (absence of clouds, relatively sparse vegetation cover). These orthorectified images appear very clear. The topographic map of Maroua 1:200 000 scale and the geological map of Leroy and Cirotteau [16] were also used.
Image Pre-Processing Method
The Landsat 8 image has undergone pre-processing, in particular radiometric correction [22] and atmospheric correction [23] to make exact overlay on topographic and geological maps, and convert the reflectances of the surface overlays to real reflectances.
Landsat Image Processing For Lineaments Mapping
Lineament mapping was carried out using digital processing from ETM+ band 3, which shows better results [24]. The principal component analysis (PCA) of the preprocessed channels makes it possible to transform the multispectral data into their principal component (PCA1). It highlights similarities and differences, thus reducing data redundancy [25]. The application of 7×7 directional filters of Sobel type (Table 1) in the NS, EW, NE-SW and NW-SE directions to the ETM+3 channels make it possible to accentuate the structural discontinuities and lithologies for a better discrimination of lineaments [26] and PCA1 makes it possible to enhance the image discontinuities corresponding to the lineaments. Automatic extraction has been used to extract lineaments because of its simplicity, speed, and it also offers high degrees of reproducibility compared to subjective manual extraction [27,28]. Only lineaments of structural origin are of interest in this study. The lineaments highlighted were analyzed and the main directions were firstly compared to those of known tectonic accidents in the region [11,13,14,17,18,19]. In order to validate the lineaments and give a structural interpretation. This step is fundamental in the validation of fractures resulting from the processing of satellite images [22]. In the second step, the lineaments extracted were compared to the lineaments of anthropic origin previously vectorized (asphalt roads, tracks, limits of forests or cultivated areas and the aligned houses) which were identified in all the lineaments and eliminated. Figure 2 shows different stages of this work which are similar to that of Rayan Gazi [29].
The lineament extraction algorithm was applied to panchromatic band 8 from geomatica software. The tuning values were selected from several tuning tests, the one that was used is recorded in Table 2.
Mapping Lineaments
Lineaments are representations of linear geological features or alignments of geological objects, topographical discontinuities or geomorphological structures inherited from ancient topographies [30,31,32]. The images resulting from the processing by directional filters made it possible to draw up the lineament maps of the study area. The one that brings out the most discontinuities is the directional filter N-S, E-W, NE-SW, NW-SE with a 7×7 matrix applied to the band OLI 3 which gives the best result ( Figure 3).
Lineaments extracted from different directional filter maps of Figure 3 allow to realize rose diagrams of A total of 3841 lineaments were obtained in the study area ( Figure 5a) and they permit to obtain the rose diagram of Figure 5b, which shows E-W (N100E) major direction and N-S (N10E) secondary direction. . S1 foliation and L1 lineation in the Kaélé region: a) S1 foliation marked by an alternation of fine discontinuous quartzo-feldspathic light beds and dark beds rich in ferromagnesian minerals in the Doumrou amphibolite; b) S1 foliation marked by the horizontal allignment of amphibole and feldspar in the Boboyo amphibolite; c and d) Plane of S1 foliation bearing mineral and L1 stretching lineation in the Boboyo and Midjivin amphibolite respectively
Field Data
The study aera was affected by polyphase deformation. Four phases of deformations are highlighted in this zone and designated D1, D2, D3 and D4 in the chronological order of their manifestation. The first phase of deformation is reported in amphibolites and is responsible of S1 foliation and L1 lineation. S1 foliation is observed in amphibolites at Doumrou, Boboyo, Kéokéo 1, Kéokéo 2, and Midjivin . In Doumrou (Figure 6a), S1 is marked by an alternation of fine discontinuous quartzo-feldspathic light beds and dark beds rich in ferromagnesian minerals in the amphibolites, while at Boboyo (Figure 6b), Kéokéo 1, Kéokéo 2 and Midjivin, S1 is materialized by horizontal allignment of amphibole, biotite and feldspar minerals in the amphibolites. The poles of the S1 are close to the center of the stereogram, consequence of their low to medium dip angles (5° to 40°). The S1 poles are located in the SE, E-ENE, W and N-NNW quadrant of the stereograms of Boboyo, Kéokéo 1, Kéokéo 2 and Midjivin respectively, results of the NW, W-WSW, E and S to SSE dip direction of the S1 foliation ( Figure 7). The L1 lineation is a composite, mineral and stretching, lineation; the mineral lineation is underlined by an alignment of amphibole and biotite, while the stretching lineation is marked by quartzo-feldspathic strips and stretched feldspar aggregates (Figure 6c and Figure 6d). In the stereogram, the poles of L1 lineation are close to the fundamental circle and concentrated around SW quadrant for the Kéokéo 1 and Boboyo stereogram, SE quadrant for the Kéokéo 2 stereogram and W to WNW quadrant for the Midjivin stereogram (Figure 7) consequence of the low dipping L1 lineation towards the SW, SE and W to WNW. This phase of deformation (D1) is strongly transposed by the second phase of deformation (D2).
The second phase of deformation (D2) is more intense and is marked by S2 foliation, L2 lineation, B2 boudin, F2 folds and C2 shear. The S2 is the result of the tectonic transposition of S1. S2 foliation is seen in metamorphic rocks: gneisses, amphibolites, banded amphibolites, quartzites, micaschists, chloritoschists, talschists. In gneisses ( Figure 8a) and banded amphibolites (Figure 8b), it is a compositional bedding with alternating millimetric quartzo-feldspathic light beds and centimetric sometimes discontinuous dark beds rich in ferromagnesian minerals. S2 foliation is mark by planer arrangement of quartzofeldspathic minerals, amphibole and/or biotite in amphibolites (Figure 8c), and quartz in quartzites. In shales (micaschist, chlorotoschist, talschist), it is marked by sheet layering (Figure 8d and Figure 8e). S2 foliation is subvertical. Its poles are located between E and S of the stereogram, reflecting W to N dipping planes at Bissélé, Gadas, Kassile, Garey, Midjivin 1, Midjivin 2 and Goubara (Figure 9a). The poles of the S2 foliation are located between N and E of the stereogram, as consequence of the S to W dip direction at Zaklang 1, Zaklang 2, Moundjouing , Aviation, Zanoumi 1, Zanoumi 2, Doumrou (Figure 9a). The poles of the S2 foliation are located between the NW and the N of the stereogram, as consequence of the SE to S dip direction at Midjivin 3 and Mazang (Figure 9a). L2 lineation is observed in amphibolites and gneisses. It is a mineral lineation marked by the alignment of amphibole and biotite in amphibolite ( Figure 8f) and a stretching lineation underlined by 8
Journal of Geosciences and Geomatics
quartzo-feldspathic straps and aggregates of stretched feldspars in gneisses (Figure 8g). L2 lineations are also observed in quartzites (Figure 8h). L2 lineation plunges towards W to NW in the localities of Aviation 1, Aviation 2, Aviation 3, Moundjouing, Zaklang 2, Zanoumi 1, Zanoumi (Figure 9b), towards SW to W in the localities of Mazang, Midjivin and Zaklang 1 (Figure 9b) and towards NE to E in the locality of Zanoumi 2. The angles of plunge are low to medium, where the positions close to the fundamental circle. Boudinage is observed in amphibolites and gneisses. It manifests as stretched syn-S2 quartzo-feldspathic materials in amphibolites (Figure 8i). Boudinage also affects amphibolites interbedded in biotite gneisses; amphibolites relatively more competent than gneiss are often cut by dextral shear into boudins (Figure 8j). These sigmoid shaped boudins are oriented N-S and NNW-SSE. They have variable dimensions 24 cm to 45 cm long axis and 12 cm to 20 cm short axis. F2 folds encountered in the studied area are isopach (Figure 8k) to anisopach (Figure 8l) folds, asymmetrical with NNE to N vergence (Figure 8m). F2 fold axes have low to medium plunging angles and variable plunging directions: W to N (Boboyo 2, Boboyo 3, Zanoumi) (Figure 9b), S to WSW (Boboyo 1, Kéokéo 1, Kéokéo 2) (Figure 9b). C2 shear affecting S1 shows dextral mouvement (Figure 8n), inducing the formation of folded microlihon. C2 shear planes are filled with the same quartzo-fedspathic materials as the S2 foliation.
Journal of Geosciences and Geomatics
D3 phase of deformation is characterized by S3 foliation, C3 shear and F3 fold. S3 foliation is evidenced in the amphibole and biotite gneisses of Mazang, where it is marked by fine compositional bedding. It is an S3/C3 syn-shear foliation, perpendicular to the S2 foliation (Figure 10a and Figure 10b). In the syn-tectonic granite, S3 foliation is materialized by planar the alignment of amphibole, biotite and feldspars (Figure 10c) and it intersects the S2 foliation in the Mazang amphibolite with approximately an angle of 30° (Figure 10d). The poles of S3 foliation are located between E and SE of the stereogram, as consequence of W to NW dip direction at Zaklang 1, Zaklang 2, Mazang and between the N of the stereogram, a consequence of the S dips at Aviation (Figure 11). F3 folds are asymmetrical folds with NE vergence (Figure 10e . F3 folds are also found as intrafolial folds ( Figure 10i) and sheath fold (Figure 10j). F3 folds are often induced by C3 conjugate shear planes (Figure 10k). F3 fold axes ( Figure 11) have a low to medium angle plunge toward NNW to N and SE to SSE (Bissélé 1), WNW to NW (Zaklang 1), NW to WNW (Zaklang 2), SW (Mazang), SW to W (Midjivin). C3 shear affects the S2 foliation. It is represented by a shear zone at Zanoumi. C3 shear is highlighted by the relative movements of the compartments that they separate and causes the folding of the S2 foliation. The movement is induced the shrinking and thickening of the S2 foliation in the shear corridor and it causes the boudinage by stretching of the quartzo-feldspathic materials of the S2 foliation. The cusp and shift highlight the dextral character of the C3 shear (Figure 10a, b, k and l). Figure 13) can be grouped into three types according to the major directions: the first type, with a major direction N120E to N130E, consists of the Lara and Mazang rose diagram with NNE-SSW (N30E to N40E) and ENE-WSW (N60E to N70E) secondary directions. The second type with E-W (N90E-N110E) major direction is made up of Kaélé and Kassilé on rose diagram. They have as secondary directions N20E to N40E, N160E (Kaélé) and N140E (Kassilé). The third type of rose diagram is that of Boboyo with the major direction of N50E and the secondary directions N70E and N160E. Unfilled joints are represented by faults and joints. Faulds in the study area are sinistral, offseting veins ( Figure 12e) or dextral, some of which are occupied by magmatic material (Figure 12b). Joints in the study area allow to realize rose diagram of Figure 13. Three types of rose diagram can be considered, according to their major and or secondary direction: (1) rose diagram with N-S major direction and variable secondary direction (Figure 13: Boboyo, Zanoumi 1, Bissélé, Kaélé, Tibiri, Mazang); (2) rose diagram with NNW-SSE secondary direction and variable major direction (Figure 13: Lara 1 and Kassilé); and (3) rose diagram with NNE-SSW major direction and variable secondary direction (Figure 13).
Deformation Regime
D1 deformational phase is characterized by S1 foliation and L1 lineation which are booth subhoriontal. The synthetic stereogram of the first phase of deformation ( Figure 14) shows that the poles of S1 are located between NNW and S and the W and WSW of the stereogram, a result of SSW to N and E to ENE of S1. The L2 lineation poles are located to SE of the stereogram, and between WNW and SSW, reflecting their plunge direction. The tangential character of these structures suggest, despite their relictual appearance on field that they have been probably affected by an horizontal general flattening. Such tangential structures (S1, L1) have been recorded in the Poli region at about 150Km south west wand is the study area [33,34,35]. Subhorizontal structures in Yaoundé area been interpreted as related to nappe stacking phase during prograde metamorphisme [36], under simple shear regime [37]. Nevertheless in the study area, no nappe formation has been yet evidenced. It is not easy to characterize the geometry of the strain during the D1 phase, because of the overprinting of D2 deformation. Never the less, it is not unlikely, that the simple shear was dominated. The second phase of deformation set up the subvertical S2 foliation carrying the L2 composite lineation (mineral and stretching), sigmoide shaped B2 boudin, dextral C2 shear which intersects the S1 foliation and the isopach to anisopach F2 folds. The fact that the L2 lineation is subhorizontal on most outcrops shows that the subvertical S2 foliation behaves like shear planes with a strong horizontal component. The asymmetry nature of F2 fold and vertical dip of S2 foliation also shows the shearing character of the second deformation phase D2. This phase of deformation is similar to D2 deformation phase in the regions of Meiganga and Doua-Kalaldi-Badzer respectively [38,39]; Nga Mbappé and Yoro north of the Yaoundé group [40]; the Tcholliré region [41]; Baïbokoum-Touboro-Ngaoundaye region [42] and also interpreted as having a shearing character. S2 foliation, L2 lineation and C2 shear are syn-migmatitic. In the stereonet (Figure 15a), we note the opposite plunge direction of P2 fold axis, globaly toward north and south. This emply a folding of F2 fold axis related to a N-S schortening. It means that the intermediate stress σ2 is also a compressional stress. The distribution of S1 pole density on a stereonet (Figure 15a) highlight a regional cylindrical F2 fold due to a main stress σ1 (N53E10SW) induce by a NE-SW compression; the intermediate stress σ2 is horizontal oriented W-E (N142E02SE) and the minor stress σ3 (N40E80NE) is subvertical. Some L2 lineation are subparallel to F2 fold axis when they should normaly be perpendicular. A rotational movement should have affected the two structures and bringing them closer one to each other. The deformation regime is a constriction, because σ1˃σ2˃σ3, but σ2 also correspond to a shortening direction. The resulting stress ellipsoid is shown in Figure 15b, characterized by an elongated triaxial ellipsoid.
The third phase of deformation (D3) is responsible of subvertical S3/C3foliation, asymetrical and sheath folds. Those structure highlight the shearing character of the D3 phase. C3 and S3 are filled by the some leucosome. The distribution of S2 pole in a stereogram defines a regional scale F3 fold (Figure 16a). This fold is due to a NNW-SSE compression induced by major stress σ1 oriented N176E14N. The attitude of regional fold axis is N67E52WSW and close similar to the mesoscale fold axis identified on the field. It is also possible that F3 regional fold being a sheath fold; in fact S2 pole tend to be distribute on a circular curve in the stereogram (Figure 16a). Mesoscale fold axis show opposite plunge direction (Figure 16a) at Bissélé, synonimous of the shortening in their direction (σ2); thus, the regime of D3 deformation phase is a constriction. Such observation has been made in the region of Baïbokoum-Touboro-Ngaoundaye [42] and in the Bafia region [40,43,44]. The result stress ellipsoid of D3 deformation phase is show in Figure 16b. Two group of F3 fold axis can be distinguished in the stereonet (Figure 16a). One group illustrating a NNW-SSE shortening (grey domain) on the other illustrating N-S shortening (yellow domain). It is possible that the direction of compression was not fixed during D3, moving from NNW-SSE to NW-SE. Figure 15. a) Highlighting the F2 regional fold from the S1 compared to the poles of L2 lineation and F3 fold axis (1. poles of L2 lineation; 2. poles of F2 fold axes, 3. Pole of regional F2 fold); b) deformation ellipsoid resulting to second phase of deformation Figure 16. a) Highlighting the F3 regional fold from the S2 compared to the P3 fold axies (1. poles of S2 foliation; 2. poles of P2 fold axes; 3. Pole of regional F3 fold; 4. regional sheath fold); b) deformation ellipsoid resulting to third phase of deformation Major directions of fracturation during D4 ( Figure 13) phase are similar to those know in Central Africa: Adamaoua shear zone ENE-WSW (N70E to N80E) [45]; NNE-SSW Cameroon Hot Line (N20-30E) [46,47,48,49]; NNE-SSW the structuration of the North domain of the CPAFBC [50]; mean direction of the Tcholliré fault (N50E) [1,51]. In Chad, the direction NNE-SSW (N30E) corresponds to the Tibesti fault [52]. The major direction N130E corresponds to the directions of the faults which border the Cretaceous series of Lamé to the south of Pala [19] and those which limit the massif of Yadé to the NW with the sediments of the Doba basin [15]. This direction also approaches the direction of the Cretaceous through of Bénoué (N135E).
Structural Significance of Lineaments
The NS, NNE-SSW, NE-SW, SSE-NNW directions of the lineaments are similar to the directions of the S2 foliations measured on the field (Figure 4; Figure 5 and Figure 13). Similar foliation directions were obtained in the Pan-African domain of Mayo-Kebbi in southwestern Chad [11,14,15,33]. These results are also consistent with data from [11,17,33,34,53] and could be due to the North-South orientation of the NW Cameroon domain. The similarities of the directions of the lineaments and of the foliations show that it is not certain lineaments could be the trajectories foliation. Such observations have been made by [54] in the region of Ngoura East Cameroon colomines and interpreted as foliation trajectories. It highlights penetrating lineaments (foliation) and nonpenetrating lineaments (fractures). The NS and EW directions of the lineaments are similar to the directions of the veins and joints in our study area ( Figure 13). Such observations were made by the analysis of spatial images which highlighted dextral E-W strike-slips in the Mayo-Kébbi domain, in southwestern Chad [17].
Structural Map
Structural map of Kaélé region is shown in Figure 17. Extrapolation of trajectories of S2 foliation highlight a regional F3 fold. Axial plane trace of this fold is oriented NE-SW and is subparallel to some regional fractures. This regional F3 fold confirm the NNW-SSE compression during the D3 phase. This result is in conformity with mesoscale fold observed on the field (Figure 16a).
Conclusion
The objective of this work was to make a structural study of the Kaéle region from landsat 8 OLI/TIRS images and field data. Image processing allows to identify lineaments in the studied area, some of which appear, in a structural map, as axial plan of regional fold. Field study permit to describe ductile and brittle structures which analyses led to decipher and group them into three successive ductile deformational phases (D1, D2, D3) and one brittle deformational phase (D4). D1 deformational phase is tangential and the regime of deformation is a horizontal general flattening. D2 and D3 are synmigmatitic phases, dominated by stretching and shearing characters, with subvertical foliations, sheat folds and subhorizontal lineations. Stress orientation change during these two phases from NNE-SSW, for the principal main stress, during D2 to NW-SE during D3. D2 and D3 deformational phases took place under constrictive regimes. D4 deformational phase shows fractures which main directions are known in the Central Africa Fold Belt. Kaélé area underwent complex structural history during which one note change of the regime of deformation and variation of the stress orientation. Future works will consist in evaluating the metallogenic potential of the study area in relation to tectonic. | 2023-02-09T16:05:14.006Z | 2023-02-06T00:00:00.000 | {
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53699437 | pes2o/s2orc | v3-fos-license | Ethnic Difference in Brain Weight of 17-20 Year-Old Males in Northern Iran
Cranial capacity and brain weight are important measurements in the study of racial/ethnic differences. Using linear (Lee–Pearson’s) formula, brain weight and cranial capacity were estimated in 398 normal 17 to 20-year-old males (200 nat ive Fars and 198 Turkman) males in Northern Iran. The dimensions of the head measured with spreading caliper and auricular head spanner. The mean±S.D of brain weight and cranial capacity in native Fars males were 1343.45 ±102.37 cm, and 1390.47 ±105.95 g, and that of Turkmans were 1163.02 ±115.76 cm and 1203.73 ±119.81 g, respectively. Cerebral Index was 3.40 ±0.37 % and 2.52 ±0.37 % in Native Fars and Turkmans, respectively and cerebral quotient was higher in Turkmans (8.34) than Native Fars males (7.95). This study s howed, the effect of ethnic factor influences the brain weight of 17-20 year-old males in Northern Iran.
INTRODUCTION
The dimensions of the head and face are measured by Cephalometry which is one of the important tools in human anthropometry (Grau et al., 2001;El-Feghi et al., 2004).These dimensions are affected by geographical, racial, ethnical, sex and age factors (Williams & Bannister et al., 1995;Golalipour et al., 2003;Okupe et al., 1984;Irmak et al., 2004).
Cranial capacity which is in close correlation with brain volume reflects racial characteristics and thus has been thought to be one of the most common items in physical anthropological studies (Von Bonin, 1934;Hwang et al., 1995).The studies using different methods on the different samples reveal the same strong pattern.These methods apply measuring brain size using MRI, endocranial volume measured from empty skulls, wet brain weight at autopsy, and external head size measurements which all of them produce the same results (Dekaban, 1978;Peters et al., 2000;Svennerholm et al., 1997;Mayhew & Olsen., 1991;Cotter et al., 1999;Nooranipoor & Masteri Farahani, 2008).
Regarding to the lack of documented studies and also the importance of acquiring information on brain weight in male living subjects in order to obtain baseline data, this study was carried out on living subjects to determine the linear dimensions of the heads measured by a classic cephalometry method as a baseline study for determining the brain weight and the effect of ethnic factor on normal 17-20 years-old males in Northern Iran.
MATERIAL AND METHOD
Three hundred ninety eight healthy 17-20 years old males of Turkman group (200 native Fars 198 Turkmans) in Gorgan, South-East Caspian Sea border (North of Iran) were designated for this study.
Turkman population has been living in this area for more than two centuries, having immigrated from central Asia.They are exclusively marring in intra-group because of religious and ethnic beliefs so are a nearly pure ethnic group.
The native Fars group are the main and original inhabitants of the Gorgan region who had been selected from among the last three generations living in this area.
Body weight and height were determined in each case.Body weights were measured by spring weighingmachine (Soehnle, Germany) while the individual swearing light clothes, with an accuracy of 1kg for weight and 1cm for height.
At the same time in each individual the following linear dimensions of the head were measured: 1. Maximum head length (L) (Glabella inion length).
Maximum head breath (B) (measured between parietal eminences).
One and two parameters measured with a spreading caliper.
3. Auricular height (HT) (external acoustic meatus to the highest point of the vertex) using an auricular head spanner.
Each measurement was taken to the nearest millimeter at least three times and the average was considered for computation.
The cranial capacity was calculated using the following formula given by Williams & Bannister et al., and Manjunath, (2002b).
Brain weight in grams and cerebral index (CI) were determined by the following formulas: brain weight = cranial capacity X 1.035.
The data for each person was recorded on a special form and then analyzed by Epi6 and comparison of the means of anthropometric measurements by T student Test (α=0.05) was used.
RESULTS
The Mean ± SD of Head Length and width and also auricular height in Native Fars and Turkman groups are depicted in Table I so that Head Length and width in Turkmans were more than that of Native Fars males but auricular height in the former was less than the latter.
Cerebral quotient was significantly higher in Turkmans (8.34)
DISCUSSION
In this study, cranial capacity of the Turkman was higher than native Fars groups.Other researchers such as Manjunath (2002a), and Hwang et al. indicated cranial capacity in Indian, Korean male populations.Also Harvey et al. (1994) showed that 41 Africans and West Indians had a smaller average brain volume than that of 67 Caucasians.
In another study Morton (1849) using the method of measuring endocranial volume on 1000 skulls with packing material found that Blacks cranial capacity averaged about 5 in 3 less than Whites.More recently, 3.40 ±0.37 2.52 ±0.37 Beals et al. (1984) carried out a large study on the endocranial volume, with measurements of up to 20,000 skulls from all around the world.He reported that East Asians, Europeans, and Africans averaged cranial volumes of 1415, 1362, and 1268 cm 3 respectively and the skulls from East Asia were 3 in 3 larger than those from Europe, which in turn were 5 in 3 larger than those from Africa.Broca (1873) corroborated the Black-White difference using endocranial volume and also found that East Asians averaged larger cranial capacities than Whites.
Also in this study, brain weight of Turkman populations (1470.33 g) was higher than native Fars males (1417.30g) so that according to our findings, the brain weight was more than Nooranipoor and Masteri Farahani study results.In their study which was done on 320 18-22 year-old males in Tehran, the center of Iran, the brain weight reported 1390.47 g.Also Hartman-Ramseir et al., (1994) reported that the mean brain weight was 1336 g in adult males with using brain autopsy method.On the other hand, Broca reported that Whites averaged heavier brains than Blacks with more complex convolutions and larger frontal lobes using the method of weighting brains at autopsy.Subsequent other studies have found an average Black-White difference of about 100 g (Bean, 1906;Mall, 1909;Pearl, 1934;Vint, 1934).
Furthermore, some studies have reported that the more White admixture (judged independently from skin color), the greater the average brain weight in Blacks (Bean; Mall; Pearl) and similarly Ho et al., (1980) determined in an autopsy study of 1261 American adults, that 811White Americans averaged 1323 g and 450 Black Americans averaged 1223 g, a difference of 100 g.Since the Blacks and Whites of the study were similar in body size, differences in body size cannot explain away the differences in brain weight.The overall size of the human brain (determined by weight) differs by almost 30% among normal subjects (Pakkenberg & Voigt, 1964;Dekaban.In this study Cerebral quotient was 8.34 and 7.95 in Turkmans and Native Fars males, respectively which was higher than the findings of Nooranipooe & Farahani's (2008) study (7.75).
Cerebral quotient = brain volume/height", which can be useful for anthropometric and clinical applications and also the pattern of the relation of brain volume and height is less affected by gender in comparison with brain weight and body weight's relation, as well.
In addition, unlike body weight, height of an individual is not affected by being underweight, overweight, or obese (Nooranipoor & Masteri Farahani).
Genetic and racial/ethnic characteristics and environmental factors can affect brain weight and cranial capacity.Regarding the genetic and racial/ethnic factor, anthropometric parameters such as cranial capacity have been shown to depend on gene expression (Okupe et al.,).In addition, different racial and ethnic groups may exhibit different patterns of gene expression.Therefore, Gene expression may be used as an important determining factor.
Several decades ago, Hooton (1926) reported that racial characteristics are best defined in the skull.Indeed, cranial capacity and subsequently brain weight constitutes one of the most important parameters for determining racial differences.Also, Okupe reported the higher fetal biparietal diameter in Nigerian than Europeans.Indeed, our previous studies revealed the important role of ethnicity in head dimentions (Golalipour et al.,;Golalipour & Hosseinpour, 2006).
Table I .
Cranial capacity, brain weight, head length and width and also auricular height in 17-20 years old Native Fars and Turkman males in Northern Iran.TableIICranial capacity, brain weight and cerebral index in 17-20 years old Native Fars and Turkman Males in Northern Iran.Ethnic difference in brain weight of 17-20 old males in Northern Iran.Int.J.Morphol., 34(3):986-989, 2016. | 2018-11-16T16:42:51.425Z | 2016-09-01T00:00:00.000 | {
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266394081 | pes2o/s2orc | v3-fos-license | Late pre-Hispanic fog oasis settlements and long-term human occupation on the Peruvian central coast from satellite imagery
Abstract Fog oases (lomas) present pockets of verdant vegetation within the arid coastal desert of Andean South America and archaeological excavation within some of the oases has revealed a long history of human exploitation of these landscapes. Yet lomas settlements are under-represented in archaeological datasets due to their tendency to be located in remote inter-valley areas. Here, the authors employ satellite imagery survey to map the locations of anthropogenic surface features along the central Peruvian coast. They observe two categories of archaeological features, large corrals and clustered structures, and document a concentration of settlement features within lomas landscapes that suggests a pre-Hispanic preference for both short- and long-term occupation of these verdant oases.
Introduction
Lomas (fog oases) are vegetation islands along the Peruvian coastal desert belt, fed by dense fog that results from the interaction between warm moist air from the Pacific Ocean and cooler, drier air currents over the Andean cordillera.The presence of archaeological sites in the lomas has led them to figure prominently in interpretations of past human-environment relationships, from early human adaptations through the development of dense populations and complex societies.Despite this research, in many ways--and especially for the later pre-Hispanic periods--lomas are treated as complementary resource zones for coastal and highland economies rather than as horizontally interconnected ecosystems (Lanning 1967;MacNiesh et al. 1975;Moseley 1975;Rick 1988;Makowski 2002;Canziani 2009;Prieto & Sandweiss 2020).
Pedestrian archaeological surveys on the central Peruvian coast have focused on coastal valleys and have paid less attention to the comparatively remote inter-valley areas where the majority of lomas ecosystems are located.As a result, lomas sites tend to be under-represented in survey datasets, and many archaeologists have devoted little consideration to them in models of the Indigenous political economies of the late pre-Hispanic central Peruvian coast.Moreover, because archaeological studies of these areas have focused on a handful of individual sites, it is unclear how representative our existing characterisations are of lomas occupations across the central coast region.
Here, we analyse data collected via visual survey of satellite imagery using GeoPACHA (Geospatial Platform for Andean Culture, History and Archaeology), and we offer a pan-regional view of the scale and intensity of human relationships with lomas in the late pre-Hispanic (c.AD 1100-1532) and early Spanish colonial (c.AD 1532-1600) periods (Table 1).Through this approach, we characterise lomas occupations at a scale that would be difficult to achieve using traditional field-based survey methods.Satellite imagery survey is especially well suited to this task, due to the high visibility of lomas sites on the hyper-arid Andean coast (Figure 1).
Our study documents evidence which extends the limited implications of small-scale studies of the nature, extent and functions of lomas occupations.Specifically, our findings propose a hypothesis that a humid period in the late pre-Hispanic period fostered permanent occupation within these ecosystems and that this occupation continued until early colonial times (Makowski 2002;Cogorno 2005;Kalicki et al. 2014).Our analyses also provide evidence to propose and discuss five additional hypotheses: 1) that there was a distinct pattern of settlement around lomas ecosystems in the north of the Rimac Valley, in comparison to areas to the south; 2) that lomas were likely not occupied simultaneously by highland herders and coastal settlers in the later pre-Hispanic period (cf.Kalicki et al. 2013Kalicki et al. , 2014;;Kalicki 2014), but rather by coastal peoples (following Mujica 1997); 3) that this pattern of coastal occupations later gave way to seasonal herding by highland populations; 4) that human occupations of the lomas ecosystem were concentrated in core areas of highest humidity and densest concentration of perennial flora; and 5) that these core areas have remained quite stable through time, persisting into the twenty-first century AD.These hypotheses arise from the pan-regional perspective afforded by systematic imagery survey but rigorous testing is not currently possible through this digital platform due to the chronological limitations of imagery-based survey.Our hypotheses are presented therefore as bases for further investigation through complementary field-based research methods.
Long-term trajectories: humans and their landscapes on the Peruvian central coast
The lomas ecosystem is the product of the interaction between marine currents, winds, Andean topography and the El Niño Southern Oscillation (ENSO) (Moat et al. 2021).Today, these verdant island niches appear seasonally between the months of July and September when atmospheric humidity is at its peak.Ecologists and archaeologists have described lomas vegetation as a key seasonal resource for both coastal and highland populations in pre-Hispanic, historic and contemporary time periods, as well as an important context of interaction between highland and coastal groups (Patterson & Lanning 1964;Lynch 1975;Kalicki 2014;Nieuwland & Mamani 2017;Baitzel & Rivera 2019).Herbaceous and woody plants useful for fuel, medicinal applications and dyes, as well as the wild ancestors of several cultigens (e.g.potato, tomato and squash) grow within the lomas (Dillon et al. 2011;Beresford-Jones et al. 2015).Lomas also act as corridors for diverse wildlife, including wild camelids (particularly guanaco), desert fox, condor and the pampas cat (Moat et al. 2021).Humans have long recognised and exploited these rich ecosystems; indeed, the archaeological record attests to human occupation of lomas from pre-ceramic times (c.4500-2000 BP) until the present day, and evidence of seasonal hunter-gatherer camps and cemeteries as well as sedentary populations in the later pre-Hispanic sequence has been recovered (Engel 1973;Mujica 1997;Makowski 2009;Beresford-Jones et al. 2015).
Beresford-Jones and colleagues (2015, 2021: 401) have, however, pointed out that the relationship between fog and lomas vegetation also varies with the ENSO cycle.The ENSO encompasses fluctuations between periods in which sea surface temperatures rise (colloquially termed El Niño) and others in which sea surface temperatures fall (La Niña).In coastal Peru, La Niña events are generally associated with reduced precipitation but more fog nearer to the shoreline (Beresford-Jones et al. 2021).La Niña events can thus bring changes in lomas vegetation patterns, favouring transitional areas near the shoreline, and limiting species of herbaceous annuals in core areas of the lomas (Pinto et al. 2001: 294;Beresford-Jones et al. 2021: 401).In the past, these oscillations could have precipitated shortterm changes in subsistence strategies and settlement patterns among lomas populations.
We define the 'core' lomas as areas with perennial woody vegetation and a wide diversity of plant species, generally found between 500m and 800m above sea level (asl).'Intermediate' areas possess a more limited range of plant species, dominated by the epiphytic Tillandsia genus, generally found between 230 and 500m asl.Finally, we define 'ephemeral' lomas as areas of vegetation that appear only sporadically, on an interannual basis, in the 100-230m elevation belt (Moat et al. 2021).
As Engel (1973: 271) observed half a century ago, there is an almost continuous distribution of lomas in a 100-500m interfluvial elevation belt between the Huacho and Cañete Valleys (i.e. the central coast region, as defined here).By contrast, to the north of the Huacho Valley lomas are scarce, and to the south of the Cañete valley they are largely confined to isolated patches separated by broad drainages that feed into the Pacific Ocean (Engel 1973;Beresford-Jones et al. 2021: 388) (Figure 2).The survey area discussed here encompasses a relatively stable and homogeneous climatic zone in terms of humidity and temperature (León 2022: 66).
It has been proposed that the Rímac and Lurín valleys comprise an intermediate zone that lies between distinctive lomas ecological regimes to the north and south (Kosok 1965: 182).In the south, between the Rimac and Cañete Valleys, lomas run roughly parallel to the coastline and encompass intermittent fluvial ravines.To the north, river courses neither parallel each other nor run perpendicular to the shoreline, and so northern lomas are distributed as more isolated green patches.
Archaeologists tended to see lomas resources as serving seasonal or marginal roles in both highland and coastal economies--that is, as sources of supplementary foods for coastal populations who focused on agriculture and marine exploitation, or as seasonal pastures exploited by transhumant highland camelid herders (e.g.Patterson & Lanning 1964;Engel 1973;MacNiesh et al. 1975;Quilter & Stocker 1983).More recently, some scholars have suggested that relationships between lomas and coastal and highland populations might be explained by John Murra's (1972) model of ecological complementarity, in which exchange of resources between different ecological zones is mediated not by markets but by kinship networks (Kalicki 2014: 85).Other scholarship has drawn attention to variability in lomas occupation through time, particularly in relation to changing climactic conditions.Kalicki and colleagues (Kalicki 2014;Kalicki et al. 2014;Kalicki & Kalicki 2020) have argued that more permanent occupation of lomas correlates with periods of higher precipitation, some linked to ENSO frequency (Kalicki et al. 2014: 157).They contend that lomas occupation was seasonal during the first half of the Late Intermediate Period (LIP) (c.AD 1000-1200), a particularly dry phase in the sequence.In the second half of the LIP (c.AD 1200-1450), this drier than average regime shifted to one of markedly increased precipitation coeval with evidence of highland population incursions (Kalicki 2014: 91;Kalicki et al. 2014: 158).Subsequently, during the Inka and Spanish periods, the introduction of foreign technologies and pathogens, exploitative political economies and renewed drier climatic conditions put an end to sustained settlement in the lomas (Kalicki 2014: 91).
An alternative interpretation posits that lomas were occupied on a year-round basis by populations that were closely linked to coastal settlements.In this model, coastal groups adapted to lomas habitation, enhanced their economic productivity and established sedentary settlements around them (Mujica 1987: 7).This 'permanent' model of coastal occupation draws, in turn, on ethnohistorian Maria Rostworowski de Diez Canseco's (1981Canseco's ( , 1989) ) argument that late pre-Hispanic coastal societies were composed of distinct, specialised economic segments that exploited specific ecosystems--in the case of the lomas, an ethno-political group called the Caringas (see also Eeckhout 2004).In turn, the Caringas were part of a larger network of coastal agricultural and fishing communities known as the Ychsma polity (Rostworowski de Diez Canseco 1981;Mujica 1997: 200).To differentiate it from Murra's emphasis on 'vertical' connections between social groups distributed across ecological tiers in the mountainous Andean landscape (Murra 1972), this interpretive focus on intra-coastal interaction is sometimes referred to as the 'Horizontal Model', and it has been extended to other regions (e.g.Shimada 1982;Zaro 2007;Pacifico & Johnson 2021).
During the late 1980s and early 1990s, Mujica and colleagues started reconnaissance of lomas ecosystems between the Lurín and Chilca valleys (Mujica 1987) and later conducted excavations in the dry quebrada of Malanche (Mujica et al. 1992;Mujica 1997).Based on this research, they proposed that year-round occupation was possible in part because of the development of complex systems of water collection and subsoil water harvesting (canals, dikes, wells) for limited agriculture activities (on terraces), complemented by foraging and herding (Mujica 1997: 208-10).In this model, herding was present in these settlements, but as a complementary activity to local occupations and was not part of highland seasonal rotation patterns.The oscillation between El Niño and La Niña events likely required diverse subsistence strategies in neighbouring ecological zones to sustain year-round occupations.Mujica proposed that the occupation of Malanche lasted from the thirteenth or fourteenth century AD until early colonial times (Mujica 1997: 203).
Scholarship by Krzysztof Makowski has contributed another level of complexity to the discussion of lomas occupations, as he argues that the Lurín Valley settlement of Pueblo Viejo was perennially settled only when it was occupied by a colony of highland mitmaqkuna, a group of forced migrants strategically resettled in the area by the Inka in the fifteenth century AD (Makowski & Oré 2013: 517;Castillo & Makowski 2019).In contraposition with the transhumant models, Makowski contends that it was only during the era of Inka imperialism that highland peoples came to occupy the site year-round, and Pueblo Viejo was abandoned following the Spanish invasion and the collapse of the Inka empire (Makowski & Oré 2013: 519).Indeed, contrary to the evidence reported at Malanche, there is no evidence of colonial occupation at Pueblo Viejo (Makowski 2002: 141).
Thus, there are two main interpretive framings of late pre-Hispanic occupations of the lomas ecosystems.One emphasises vertical interaction with lomas featuring as a peripheral zone for both coastal and highland populations, suggesting fluid interactions between these populations who each, separately, exploited these ecosystems.A second emphasises horizontal interactions among coastal groups and year-round occupations.Both approaches are based on two assumptions.First, that permanent (year-round) occupation in the area was only possible during the period of increased humidity during the first half of the LIP (c.AD 1100) through to early colonial times.And second, that lomas distributions throughout the central coast are regular and uniform; less attention is given to how ecological variation within and across lomas patches might have influenced the roles they played within regional political economies.
These assumptions are based on results from fragmentary field research; intensive archaeological research at lomas sites has focused on a very small number of locations.Findings from these locations have then been used to generate general hypotheses about lomas occupations.We argue that to properly test the validity of these interpretive frameworks new sources of data are needed which span the diversity of lomas across the central Peruvian coast, as well as adjacent regions.Large-scale imagery survey enables such a broadened perspective.
Patterns in the human occupation of lomas
The GeoPACHA project enables visualisation of high-resolution satellite imagery from multiple sources (including Google Maps, Bing Virtual Earth, ESRI high-resolution imagery and MapBox) and the systematic survey of large areas through a grid-based tracking system (see Wernke et al. 2023, for details of the GeoPACHA project).To assess variation in lomas occupation, we employed GeoPACHA to conduct a systematic satellite imagery survey of archaeological loci (features of archaeological interest) within a portion of the inter-valley zone of the Peruvian central coast.The hyper-arid characteristics of the central coast are ideal for this kind of imagery-based feature detection and we surveyed an area of almost 8800km 2 stretching from the southern edge of the Huaura Valley in the north to the northern edge of the Cañete Valley in the south (Figure 3).These boundaries are historically meaningful as they map the extent of the Lima culture of the Early Intermediate Period (c.200 BC-AD 700) and the Chancay and Ychsma societies of the Late Intermediate Period (c.AD 1100-1470) and Late Horizon (c.AD 1470-1531).We started our survey from the southernmost border of the project area and worked northwards, tagging (with a single point) each relict anthropogenic feature visible in the satellite imagery.We used an arbitrary 100m rule to define locus boundaries--that is, any feature located greater than 100m from its nearest neighbour was considered a separate locus and features less than 100m apart were grouped together as a single locus.In total, 1018 discrete archaeological loci were identified, most within a low elevation band that stretches across the inter-valley plains and foothills, extending inland about 30km from the coast.Of these, 342 loci are in areas with evidence of past or present lomas ecosystems.
For the purposes of this article, we focus on spatial patterns within the valleys, mostly in the foothill areas.The concentration of loci within the foothills at the same altitude that lomas are typically found would seem to suggest that these sites represent the remains of lomas occupations.To test this observation, loci locations were compared with lomas distribution mapped by Moat and colleagues (2021), based on analysis of 20 years of MODIS satellite imagery (2000-2020) and complemented with assessment of vegetation productivity through the Normalised Difference Vegetation Index (an estimation of the density of green coverage within an area; Figure 4).Moat and colleagues mapped 17 000km 2 of extant lomas Late pre-Hispanic fog oasis settlements and categorised them using six classes.Class 1 represented ephemeral lomas while class 6 represented core areas of highest and most sustained humidity with herbaceous and woody land cover.Multiple lines of evidence--including lithic flux in marine sediments (Rein et al. 2005) and proxies in sediment cores from Laguna Paucallcocha (Moy et al. 2002)-indicate a period of higher humidity in the central coast region during the early LIP, linked to intensified El Niño events (Kalicki 2014: 88).Archaeologists have struggled to relate this climactic pattern to changes in settlement patterns, however, and it has been unclear if settlement distribution expanded during this period and/or if any changes arose in the seasonality of local occupations.To assess whether pertinent patterns may be discerned within our data, we compared the distribution of archaeological loci identified in GeoPACHA with the extent of six classes of lomas ecosystem identified by Moat and colleagues (2021).
A Monte Carlo simulation was used to test whether the observed distribution of loci was significantly different from a random distribution with respect to the lomas categories proposed by Moat and colleagues.This simulation repeatedly distributes points across the study region and compares the randomly generated distribution to the observed distribution of loci.The null hypothesis, that there is no relationship between the location of loci and lomas persistence within the survey zone, must be accepted if the randomly generated points are distributed between areas of different lomas classes at approximately the same rates as the recorded loci.If the distribution is concentrated in a particular area, the null hypothesis will be rejected, and the relation between loci and specific class in Moat et al. distribution proved.
Results
Analysis of the distribution of archaeological loci identified in the survey area demonstrates notable differences between those lomas areas located south of the Rimac Valley and those located to the north (Figure 2).Within the 4265km 2 area surveyed south of the Rimac Valley, 1768km 2 of lomas ecosystem was mapped and 243 loci were identified within the lomas.North of the Rimac Valley, 1438km 2 of the 4535km 2 area surveyed was identified as lomas ecosystem and contained 101 loci.This difference may relate to the lomas distribution itself and to variations in the geomorphology of the coastal belt.North of the Rimac Valley, lomas are present as green pockets isolated from valleys but, to the south, between the Rimac and Cañete valleys, lomas form an almost continuous vegetation band that runs parallel to the coastline and perpendicular to valleys and dry ravines (quebradas).
The survey showed that two general types of archaeological loci are found in lomas areas (Figure 5).The first are large, isolated structures we call corralónes (large corrals), which previous archaeological field research suggests were used by pastoralists as overnight pens for camelids (Mujica 1997;Kalicki 2014).These structures are easily identifiable from the satellite images as high fieldstone walls enclosing large areas (ranging between 100 and 200m 2 approximately) (Figure 6).A second type of locus is composed of agglutinated structures of different sizes, with walls in such a poor state of preservation that they are much harder to discern in satellite imagery.The poor state of preservation is likely due to their older age relative to corralónes.This second type of structure may be linked with late pre-Hispanic occupation (Figures 7 & 8).
Comparison of the distribution of archaeological loci identified through GeoPACHA with the extent of the six classes of lomas identified by Moat and colleagues (2021) shows a clear tendency for loci to be concentrated principally in areas of greater humidity (Figure 9).Table 2 presents the percentages of lomas areas comprised by each class and the percentage of loci found in each of these zones.The greatest proportion of loci identified by our survey were found in association with the areas of greatest biomass and herbaceous, woody plant cover (class 6).
To further test the significance of loci distribution, a Monte Carlo simulation was conducted with 100 000 iterations and generated a significant result with a p value of 0.00002.This indicates that only two out of the 100 000 random point patterns were as extreme, or more extreme, in their split between lomas classes than the observed pattern of loci.The null hypothesis is therefore rejected and a strong association between identified loci and the contemporary extent of lomas vegetation is proposed.This suggests that most of the core vegetation areas will have been relatively stable for some time, as they are driven by the unimodal winds and the Humboldt current (a cold-water current that flows north from Antarctica along the west coast of South America).As a result, we suggest that, in aggregation, late pre-Hispanic occupations were preferentially situated within perennial herbaceous and woody fog oases and did not become more dispersed during the early LIP.Rather, settlement location remained stable, and an intensified, year-round occupation of lomas was supported by horticulture and complementary herding.
Notes for a chronological discussion
One of the greatest limitations of satellite survey in archaeology is the inability to acquire clear chronologies in the absence of sites with distinctive features that have themselves been dated using other techniques.Fortunately, previous archaeological field research on lomas sites provide us with a means of estimating the chronologies of the loci identified in this study.
Most pre-ceramic sites (dating to before c. 1800 BC) within the lomas are seasonal camp sites, with little apparent surface architecture (e.g.Patterson & Lanning 1964;Engel 1973;Beresford-Jones et al. 2021).In the Lachay region (see Figure 2), this pattern of occupation continues more or less unchanged during the Early Horizon (c.800-200 BC), with the exception of the appearances of an isolated public building (Kalicki et al. 2014: 155).Similar dynamics are reported for the Lurín Valley, where isolated ceremonial centres serve as indicators of dispersed communities living close to their catchment areas (Burger 2014).By the end of this period, some cemeteries are also reported in the lomas (e.g.Makowski 2009).These early camp and cemetery sites are not likely to be represented in our survey.
For the subsequent Early Intermediate Phase (c.200 BC-AD 700), occupation structures composed of single-coursed walls associated with agricultural terraces are reported at Lachay (Kalicki et al. 2014: 155).In the southern part of our survey area, no Early Intermediate Phase sites have been reported in a lomas context, although several sites do exist within valleys at comparable altitudes to lomas ecosystems--for example, the site of Lote B in the Lurín valley (Marcone 2016).For the Lachay area these Early Intermediate Phase sites could have been re-occupied during the LIP as well (Kalicki et al. 2014).Therefore, although there is some suggestion of year-round occupation of the lomas in the Late Intermediate Period, lack of investment in architectural materials and the fact that many sites were later re-occupied, indicates that there is an under-representation of Early Intermediate sites within identified loci.
The Middle Horizon (c.AD 600-1000) is a period of population contraction throughout the central coast, and lomas ecosystems are no exception.No lomas sites have been reported for the Middle Horizon, with the exception of cemeteries such as Teatino in Lachay (Kalicki et al. 2014: 156).By the LIP, however, several sites are reported in the lomas (Mujica 1987(Mujica , 1997;;Kalicki 2014).These complex sites include extensive agglutinated domestic areas, storage, terraces and other architectural features which suggest year-round occupations, and are clearly identifiable from the satellite imagery.We therefore propose that most loci showing agglutinated structures identified within our survey date to the LIP.Some may also correspond to subsequent Late Horizon occupations from the fifteenth century AD.Small temporal differences (which could be less than 100 years) between the LIP, Late Horizon and early colonial era are only possible to address in relation to specific intra-site studies (e.g.Makowski 2002).These studies in general support the idea of a strategic (though indirect) Inka intrusion in the central coast, through neighbouring highland people (Makowski & Ore 2013).
The other type of structure recognised in our survey is the corralónes used for pastoralist activities.These structures are generally rebuilt on a seasonal basis, which makes them particularly easy to identify in our survey but very difficult to date.We hypothesise that most of these isolated structures are post-conquest constructions, but this initial assumption needs to be corroborated by future field research.
Discussion: some regional views of lomas ecosystem occupation The regional view provided by our survey extends the idea of a year-round occupation of the most productive and perennial core areas of lomas during the early part of the Late Intermediate Period, which corresponds with a period of increased humidity along the Peruvian coast (Moy et al. 2002;Rein et al. 2005).From our pan-regional satellite imagery survey, we propose that past human occupation of the lomas concentrated in the core areas of this ecosystem (Figure 9) and that these core areas have remained quite stable through time, persisting to the present day.
A review of field research in the survey area indicates that structures identified in our survey likely date from the LIP or later.Among the loci we were able to identify two types of structures.Those possibly relating to herding (corralónes) appear to be more recent, or at least to have been maintained more regularly, than the complexes of agglutinated structures we also identified, which were apparently abandoned prior to the appearance of the tradition of corralónes construction and remain in a poor state of preservation.
The differences in the distribution of archaeological loci we have identified in the lomas between the northern part and southern part of the central coast could help to explain why research from Lachay, in the northern extent of our survey area, supports the transhumance model, where highland herders and coastal settlers in late pre-Hispanic times shared access to lomas (Kalicki et al. 2013(Kalicki et al. , 2014;;Kalicki 2014).Research from more southern areas proposes that late pre-Hispanic occupation was permanent and in direct interaction with coastal developments (Mujica 1987(Mujica , 1997;;Makowski & Oré 2013).In this sense, Pueblo Viejo (the Inka era settlement discussed above) could be the exception that confirms the rule.The presence of highland communities in the region seems to have been facilitated by the Inka state; it was not the result of a long-term or 'organic' settlement radiation from the highlands prior to Inka rule (Mujica 1997;Makowski & Oré 2013).In this southern portion of our study area there are no purported permanent settlements of highland populations other than Pueblo Viejo.This idea is also supported by the ethnohistoric data, which suggest that southern lomas were occupied by the Caringas Ayllu, part of the Ychsma coastal society (Rostworoski de Diez Canseco 1989).
Conclusions
The study of lomas settlements through imagery survey in GeoPACHA provides regional perspectives that, when compared with previous field research, produce a series of refined hypotheses about the human occupation of the central coast of Peru.As Wernke and colleagues argue in their overview of the GeoPACHA project (Werneke et al. 2023), archaeological hypotheses concerning large-scale phenomena are often generated (and then tested) based on very limited samples.A critical first step to improving large-scale archaeological research is therefore to generate hypotheses for testing based on initial observations of large-scale systems.This article provides that first step and continues the discussion surrounding variation within the spatial distribution of lomas and hypotheses of human occupation within these ecosystems.Testing these hypotheses will require additional systematic pedestrian survey and excavations designed to sample the variation in lomas occupations identified by our satellite survey.
By highlighting an apparently long-term association between substantial settlements and perennial herbaceous and woody fog oases, this study suggests that it may be productive to think of lomas not as inveterately transitional, liminal or 'complementary' resource zones located between coastal and highland societies-at least during the Late Intermediate Period.
In this way, our large-scale imagery survey has generated both new insights and new questions for continued field investigation in both the northern and southern parts of the central Peruvian coast.As lomas ecosystems are threatened by climate change and development, these research efforts are an urgent priority for understanding the long-term history of this part of Andean South America.
Funding statement
The survey of the central coast was possible through the financial support of the Center for Impact and Social Responsibility (CIRSO) of the Universidad de Ingenieria y tecnologia (UTEC).GeoPACHA was supported by grants from NEH, ACLS, SPARC, Vanderbilt University, Brown University Dean of Faculty, and a Stanley J. Bernstein Assistant Professorship in the Social Sciences.
Figure 1 .
Figure 1.View of the lomas environment (foreground) in contrast to the coastal desert beyond (photograph by Alejandro Bryce).
Figure 3 .
Figure 3. Survey area in the central coast showing loci identified in lomas ecosystems (figure by Giancarlo Marcone).
Figure 4 .
Figure 4. Locations of archaeological loci in relation to lomas distributions.Lomas areas based on Moat et al. 2021 (figure by Giancarlo Marcone).
Figure 5 .
Figure 5. Examples of isolated structures or corralónes identified on satellite imagery (images from Google Earth 2022 CNES/Airbus; figure by Giancarlo Marcone).
Figure 6 .
Figure 6.An example of a corralón at ground level (photograph by Alejandro Bryce).
Figure 7 .
Figure 7. Satellite images of Malanche dried waterway structures, as an example of agglutinated structures identified in lomas ecosystems (images from Google Earth 2022 Maxar Technologies; figure by Giancarlo Marcone).
Figure 8 .
Figure 8. Examples of small structures at ground level during dry season (photographs by Giancarlo Marcone).
Figure 9 .
Figure 9. Distribution of loci and lomas classes.Class 6 represents lomas which persist more than 50 per cent of the year, while class 1 are ephemeral oases which are present for less than one week per year.See Table 2 for further information (figure by James Zimmer-Dauphinee).
Table 1 :
Schematic chronological table for the Peruvian Central Coast. | 2023-12-21T16:33:44.397Z | 2023-12-18T00:00:00.000 | {
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225040055 | pes2o/s2orc | v3-fos-license | Deep learning prediction of patient response time course from early data via neural-pharmacokinetic/pharmacodynamic modeling
The longitudinal analysis of patient response time course following doses of therapeutics is currently performed using Pharmacokinetic/Pharmacodynamic (PK/PD) methodologies, which requires significant human experience and expertise in the modeling of dynamical systems. By utilizing recent advancements in deep learning, we show that the governing differential equations can be learnt directly from longitudinal patient data. In particular, we propose a novel neural-PK/PD framework that combines key pharmacological principles with neural ordinary differential equations. We applied it to an analysis of drug concentration and platelet response from a clinical dataset consisting of over 600 patients. We show that the neural-PK/PD model improves upon a state-of-the-art model with respect to metrics for temporal prediction. Furthermore, by incorporating key PK/PD concepts into its architecture, the model can generalize and enable the simulations of patient responses to untested dosing regimens. These results demonstrate the potential of neural-PK/PD for automated predictive analytics of patient response time course.
Introduction
The longitudinal analysis of patient response time course following doses of therapeutics is an important topic for drug development and personalized medicine. However, this is a highly challenging task due to the variability of patient response to treatments and the complexity of drug mechanisms. In response to the need for better characterizing the dynamics between dosing, drug concentration and patient response, the discipline of Pharmacokinetic/Pharmacodynamic (PK/PD) has been developed to link systemic drug concentration kinetics (i.e., PK) to the resulting pharmacological effects over time (i.e., PD) using mathematical models [1]. These models enable the description and prediction of the time course of physiological effects (e.g. tumor size, platelet count, etc.) in response to drugs of various dosage regimens [1,2]. PK/PD models are typically developed using ordinary differential equations (ODEs), the construction of which have relied upon human modelers' abstraction of data into dynamical systems. Currently, the state-of-the-art methodology for estimating the set of model parameters in PK/PD models is the population approach [3], whereby certain statistical distributions of parameters and error models are assumed and subsequently iterative optimization techniques are applied to computationally minimize the discrepancy between observed and predicted trajectory in some appropriate error metric [4,5,2]. The performance of alternative models is compared, and a selection is ultimately made based on various diagnostic criteria and modeling statistics [4,5]. As described above, the population-PK/PD (pop-PK/PD) modeling paradigm is highly iterative in nature. The accuracy of such models for making temporal predictions depends on the modeler's ability and expertise in describing complex data sets with mathematical equations. However, as the range of data modalities increase in modern biomedical applications to include imaging, high dimensional assays, and continuous monitoring devises, it becomes ever more challenging for the human modelers to glean insight from such large volumes of data. Coupled to the growing data is the need for improving PK/PD models' ability to perform temporal extrapolations, which is key to precision dosing applications [6]. Motivated by these challenges, we explore the possibility of using deep learning to build PK/PD models to augment human capabilities in abstracting dynamical systems from patient data following drug treatment while enhancing predictive accuracy.
With the recent development of the neural ordinary differential equations (neural-ODE) methodology [7], it has become possible to consider an ODE modeling paradigm whereby one can learn the governing equations algorithmically and directly from the data [8,9]. In particular, this novel machine learning approach generates the input-to-output mapping as the numerical integration of an ODE system described by a neural network, in which the backpropagation is carried out by an adjoint solution method [7]. The neural-ODE methodology is well suited for time-series analysis and especially in the PK/PD setting, as both the dosing and measurement times can be irregular. In particular, it has been applied to fields such as the life sciences [10], image processing [11] and computational physics [12,13,14]. While there are several examples of neural-ODE methodologies that have been developed and applied to publicly available biomedical data sets (such as PhysioNet) [10,15,16], there is currently no implementation in which the dosing of a therapeutic drug and its concentration data are both explicitly represented in the model. In addition, as these model formulations do not place constraints on the form of ODE systems being constructed and lack the incorporation of key pharmacological principles into their architecture, their ability to generalize from the training set and predict unseen dosing regimens remain in question.
In this work, we propose a new deep learning approach to build PK/PD models that directly learn the governing equations from data with the aim of predicting patient response time course as well as being able to simulate the effects of unseen dosing regimens. Our approach is novel in two ways. Firstly, the architecture of our deep learning model ensures that the pharmacological principle of dose-concentration-effect is preserved; that is, the model assumes the causal relationship of dosing driving the drug concentration, which in turn drives the effect dynamics. Secondly, the network architecture is constructed in a manner such that the dosing data enters the model via not one but two ports, thereby ensuring the ability of the model to not only predict the existing treatment data but also enable the simulation of "what-if" scenarios whereby the patients' dosing regimen is modified. Our work illustrates how by incorporating key domain specific modeling principles into the neural network architecture, human and machine intelligence can work together in building dynamical systems that are more likely to generalize well beyond the training data and be better embraced by the domain experts.
Herein, we detail a sequential methodology for the construction of a neural-PK model and a neural-PK/PD model. We demonstrate the approach in describing and predicting drug concentration and platelet dynamics following trastuzumab emtansine (T-DM1) treatment, which is an approved anti-cancer therapy (intravenous administration at 3.6 mg/kg once every three weeks [Q3W]) for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer in patients failing prior treatment with trastuzumab and a taxane [17]. Thrombocytopenia, a decrease in platelets requiring dose reductions and delays, is the dose limiting toxicity for T-DM1 [17]. To show the utility of this methodology, we benchmark against the original pop-PK/PD analyses [18,17], which utilized the myelosuppression PKPD modeling approach proposed by Friberg et al [19] that is considered the "gold-standard" for modeling such clinical data. We develop a neural network to search within a space of ODE systems no larger in dimension than that of the existing pop-PK/PD model [17] and compare the resulting neural-PK/PD model with pop-PK/PD in terms of their ability to predict future platelet counts from early data at the individual patient level. Finally, we illustrate the generalizability of neural-PK/PD predictions by performing simulations to predict the effects of alternate (and untested) dosing regimens.
Overview of Model
We incorporate the basic principles of PK/PD [1] into our neural-PK/PD architecture ( Fig. 1) by encoding the following standard PK/PD assumptions: (1) the dynamics of PK is driven by dosing and is independent of PD; (2) the dynamics of PD is driven by PK as well as by itself [1]. We built the above assumptions into the computational graph of the model via the explicit representation of the "Dose" input to the ODE sub-module as shown in Fig. 1(a), and in the dependence of the PK and PD vector fields shown in Fig. 1(c) and (d). In particular, the "Dose" data enters as Dirac delta forcing to the PK component of the ODE system (refer to the Methods section and Supplementary Fig. 1(b) for further details). This precise treatment of dosing is a key part of our model implementation that follows the traditional PK/PD modeling paradigm and which helps to ensure the generalizability of the model predictions to unseen dose schedules. The PK components of the ODE vector field are not influenced by the PD states, whereas the PD components of the ODE vector field are influenced by both the PK and PD states. The individual patient data (including the observed drug concentration, dosed amounts and pharmacodynamic response) is fed into the network port "PKPDData"; see the Methods section for more details. An innovative feature of our model is that drug dosing enters the network at two locations: as part of the "PKPDData" and also through the "Dose" port. While the dosing data that forms part of "PKPDData" represents what doses patients have been treated with, the dosing that enters "Dose" port represents what the model uses in making predictions. For the purpose of reproducing the data in the training set, the doses that enter the two separate ports are identical. However, once the model has been trained and a novel dosing regimen is to be tested, the dosing data that enters the "Dose" port is modified as desired. For details on the training and the structure of the neural-PK model, please refer to the Methods section.
PK (drug concentration) data is recapitulated almost perfectly
In the current work, the PK and PD parts of the network are built in a sequential manner [20], as is the case for the original pop-PK/PD model [18,17] that we benchmark against. In particular, we take a pop-PK model that was built previously for T-DM1 [21,22] and train a neural-PK model to mimic the pop-PK model by reading in the early PK data and predict the future time points. We used T-DM1 treatment dataset involving 665 patients, with a median observation and dosing record of 169 days (minimum: 1 day, maximum: 862 days). We split the total number of available patients into a training and testing set. For details on the training and the structure of the neural-PK model, please refer to the Methods section. As illustrated in Fig. 2, the trained neural-PK model can effectively predict (on unseen test patients) the complete time-course of the pop-PK model for T-DM1 drug concentration, by using only the observed PK data up to day 21, which is the first cycle of the treatment. For a quantitative assessment of the neural-PK model, we evaluated it on the n=133 test patients with 3228 PK observations made from times t ³ 21 days. The predicted drug concentrations from the neural-PK model are in a good correspondence with the predicted drug concentrations from the original pop-PK model, with r-squared ( 2) = 0.98, correlation coefficient = 0.99 and the root-mean-squared-error (RMSE) of 2.67.
Qualitative dynamics of PD (drug response) is recapitulated
By using the trained neural-PK sub-module to drive the neural-PD sub-module, we train the neural network to generate a dynamical system that recapitulates the observed patient platelet trajectories (see the Methods section for details). Fig. 3 shows a comparison of predictions from the neural-PK/PD model to the pop-PK/PD model [17] with the actual platelet data for randomly selected example patients from the test set. In particular, the observed drug concentration and platelet data from the first dosing cycle (i.e., up to 21 days) are provided to the neural network, which subsequently generates the predicted drug concentration and platelet response for all future time points (after 21 days) based on the individual dosing information. The result shows that by incorporating PK/PD principles into its network architecture but otherwise without additional human input, the proposed methodology is able to generate a PK/PD model that demonstrates comparable qualitative dynamical behavior to the state-of-the-art model [17]. In particular, as shown in Fig. 3 the neural-PK/PD model predictions demonstrate a drop in the platelet count post dosing, the subsequent recovery dynamics, as well as a gradual decrease in both the peaks and nadirs of platelet counts in some patients who have been treated for long durations. While significant human expertise and effort was required to build a complex PK/PD model [17] (containing negative feedback, time-dependent and nonlinear terms) based on the Friberg formalism [19], a qualitatively similar dynamical system was constructed in an automated fashion by applying neural-PK/PD modeling directly to data.
Neural-PK/PD model outperforms the current "gold-standard"
We benchmark our neural-PK/PD model against the current "gold-standard" pop-PK/PD model [17]. We considered the following 3 sets of observation limits: tObs = 21, 42 and 63 days. In each case, we use the data within the initial observation window (t < tObs) in order to predict all the future platelet data (t ³ tObs). Fig. 4 presents a comparison of pop-PK/PD and neural-PK/PD model predictions versus the observed data ("ground truth"), demonstrating that the latter surpasses the former using both the r2 and RMSE measures of prediction performance. In fact, as shown in Table 1 (Cases (a) to (c)), for all observation windows (Obs = 21, 42 and 63 days) the performance of neural-PK/PD surpasses that of pop-PK/PD by a sizeable margin.
What are the potential implications of improving the prediction accuracy using neural-PK/PD modeling as compared to the current pop-PK/PD modeling approaches? We considered the following scenario: suppose we wish to predict the individual platelet counts in test patients, over the time horizon of t ³ 42 days. Using the existing pop-PK/PD approach whereby data is observed for tObs < 42 days, we obtain r2 = 0.39 and RMSE = 59.8 (refer to Case (b) in Table 1). In contrast, using the proposed neural-PK/PD approach we can make more precise predictions (r2 = 0.45, RMSE = 56.36) using only half the observation window, tObs < 21 days (refer to Case (d) in Table 1). The ability of the neural-PK/PD model to use less data while improving the precision of forecast for patients' future response as compared to the current state-of-the-art model makes it a promising methodology that could potentially enable more robust predictive analytics based on earlier observation. To our knowledge, this is the first demonstration of a deep learning model that surpasses the state-of-the-art pop-PK/PD model in terms of predictive performance.
Simulating alternative dosing regimen
One key use of pop-PK/PD model is to perform simulations of new dosing regimens of interest [4,5] and predict the corresponding drug concentration and response of such untested scenarios in patients. By design, dosing enters neural-PK/PD model as an independent input thereby ensuring that different dosing schedules can be evaluated as desired. As a demonstration of the utility of neural-PK/PD models to predict alternative dosing regimens, we show in Fig finding that is consistent with the pop-PK/PD simulation results [17]. Furthermore, the PK accumulation observed for the Q3D dosing is consistent with the expected PK behavior of T-DM1. The ability of the model to generate simulations with previously unseen dosing stems from the neural network architecture having been designed based on PK/PD principles, resulting in meaningful extrapolations.
Figure 1. Schematic diagram of the neural-PK/PD model and the vector field (VF) sub-modules. (a) The input data (PK, PD and dosing) is passed as a table of numeric values into the network port "PKPDData", which is subsequently encoded into
vectors of various dimensions along 3 parallel pathways: (1) "PKData" ® "PKEncoder" feeds into the PK sub-module and determines the PK time course; (2) "PDData" ® "PDEncoder" feeds into the PD sub-module and determines how the PD variables change with time in accordance with PK; (3) "ICNet"® "Sum" feeds in the initial condition of the ODE system. The "ODE" sub-module is a recurrent neural net that unfolds along the time dimension to produce a sequence of predictions for PK and PD. The model generated PK and PD predictions are sent to the network port "ObsState". (b) The inputs to the VF network consist of the current value of the state vector (denoted as "PrevState") as well the PK and PD parameters from the respective encoder networks. The computation of "PKVF" is performed using the PK components of the state vector and the PK parameters. The computation of "PDVF" is performed using the PD components of the state vector, the PD parameters and the drug concentration in patients' plasma "CP". The results are subsequently concatenated and sent to "VFOut". (c) "PKVF" consists of linear and "SoftPlus" [23] layers. (d) "PDVF" consists of linear and "SELU" [24] layers. The numbers in grey denote the dimensions of the tensors/vectors that are being sent. is driven by data and can be generated algorithmically with little direct human supervision thus saving time and human resources, it remains to be validated on many datasets, and we hope this study will spur such analyses in the near future. In addition, the incorporation of variability into neural-PK/PD models so as to enable clinical trial simulations remain to be further developed.
Although deep learning models have been shown to have impressive ability to directly learn from vast amounts of data and enable predictions with little human intervention, existing techniques are known to have certain drawbacks [25,26,27]. Firstly, many deep learning models tend to be data hungry [28] and their applicability to data sets of moderate size that arise from clinical trial settings may be limited. Secondly, it is known that deep learning models may use features in the training data that do not generalize well to other situations [26], thereby potentially lowering the utility of such models especially for prediction of untested scenarios. We believe that the incorporation of key domain specific concepts into deep learning frameworks is an effective way to combine human and machine intelligence and can be crucial for the successful applications of such models. Within the domain of longitudinal analysis of patient treatment data, we propose the incorporation of wellestablished PK/PD principles into neural-ODE framework [7] by an appropriate choice of network architecture as a way to ensure that these models would serve well for the purpose of individualized predictions of the effects of untested dosing regimen. We demonstrate the feasibility of such novel approach for drug concentration and response prediction based on a legacy clinical trial data set consisting of more than 600 patients. While the results shown in this work demonstrate the proposed neural-PK/PD modeling approach can improve upon the current "gold-standard" pop-PK/PD modeling on prediction metrics, the clinical implications and meaningfulness of such improvements need to be further assessed in future work. The importance of using machine to aid human to build models will increase as technological advancements lead to an ever-increasing set of diagnostic modalities and monitoring devices that generate growing amounts of patient treatment data in real time; relying on human insight alone to generate models that describe the observed data will become increasingly challenging in the digital age. With the joint requirements of automating modeling to save time and human resources, while ensuring the pharmacological meaningfulness and the predictability of the generated models to inform dosing considerations, it is important to bridge modern deep learning and traditional PK/PD methodologies. Although machine learning is currently not part of clinical pharmacology modeling applications, it is appreciated that a number of opportunities exist for leveraging both paradigms [29]. We propose that the "artificial intelligence (AI) enabled clinical pharmacologists" of the future [30] would tap into neural-PK/PD as one of the advanced analytics tools to understand and predict drug concentration and response for dosing recommendation. Additionally, as neural-PK/PD models "speak" the language of neural networks, it could be combined with other deep learning models for novel data types and can as well be integrated into digital devices for automated longitudinal patient monitoring to generate treatment insights.
While the proposed neural-PK/PD formulation demonstrates encouraging results, there are several areas that remain topics for future work. The current model in the form of a recurrent architecture that takes fixed time steps in the numerical approximation of the underlying ODE system. Various efforts are underway to generalize the current framework by incorporating neural ODE solvers that take adaptive step sizes (e.g., [7]), so as to account for the arbitrary time values that may be present in the data set (i.e., measurement and dosing times). While the data set shown in this work involves time points that take on integer values (in the unit of day) and hence directly applicable to the proposed model, the incorporation of adaptive step size is expected to provide increased efficiency and accuracy in the general setting. For this extension, although the "ODE" sub-module shown in Fig. 1 would need to be replaced by an adaptive ODE solver [7], the architecture of the proposed neural-PK/PD model would remain the same. Finally, in the current work we did not use any baseline features of the patients (such as demographics, pre-treatment lab values, etc) for a head-to-head comparison with the original pop-PK/PD model [17] which similarly did not include any covariate effects.
Incorporation of the available baseline features may better characterize patient heterogeneity and improve the model predictivity, remains a topic for future work. In general, this novel neural-PKPD approach also need to be tested with more drugs, indications, as well as various type of therapeutic and adverse responses, to further support the robustness and predictivity of the approach in clinical settings. To conclude, the promising results shown in this work and the potential advantages offered by this novel neural-PK/PD methodology warrants further development and testing. We expect that the development and deployment of neural-PK/PD models would help to facilitate clinical drug development and advance personalized medicine in clinical practice.
Methods
In this work, the platelet count measured in patients' blood corresponds to the PD variable whose time course we attempt to describe in relation to the PK (or drug concentration). We compare the neural-PK/PD model with pop-PK/PD in terms of their ability to predict future platelet counts from early data. We do so by taking 80% of the total patients in the data as the training set and the remaining 20% as the test set, in a manner following the machine learning paradigm [31,32]. In particular, we consider a hypothetical scenario whereby we have obtained data from a prior clinical study (i.e., training set) and built a model on it; subsequently, we would like to apply the model to a new trial of a similar patient population and predict the individual patient future platelet time course from having observed only the early data. While pop-PK/PD models are typically evaluated based on their ability to describe the available data with parsimony considerations [4,5] rather than for their ability to make temporal prediction, the current approach is one way to compare the models on an equal footing.
Data
The data consists of longitudinal platelet response from 665 patients receiving T-DM1, including patients from one Phase I study (TDM3569g), three Phase II studies (TDM4258g, TDM4374g and TDM4450g) and one Phase III study (TDM4370g). Please refer to [18,17,21] for further details regarding the clinical studies and the patient demographics. All patients
Data processing for neural network
From the entire data set, an 80%-20% split of patients was made to divide them into the training (n=532) and testing (n=133) subsets. Data normalization was performed by taking the complete time course from the training set, and the normalization factors are computed so as to ensure each variable (i.e., data column) is normalized to have mean of 0 and standard deviation of 1; subsequently, the same scaling factors are applied to transform the testing set.
To combat overfitting, data augmentation on the set of training patients was performed as follows: we take the union of the following data sets, where in each case the notation "input" → "output" denotes that the network is fed "input" into the network port "PKPDData" and asked to predict the "output" as the prediction target:
Neural-PK model and training
The neural-PK model was constructed to reproduce the T-DM1 concentration time-course predicted by the pop-PK model [18]. The schematic diagram of the model is shown in Supplementary Fig. 1(a). The input data enters the network through the port "PKPDData"; the data consists of a variable number of rows (denoted by n1 in Supplementary Fig. 1(a)) and 5 columns, which are: (1) time-after-dose, (2) time, (3) PK, (4) platelet count, and (5) amount dosed. In particular, for our neural-PK model, the platelet count is not used under the assumption that PK is not influenced by PD. Hence, only the columns 1, 2, 3 and 5 are selected via the "PKPart" network. The selected data columns are then fed into the "PKEncoder", which at its core contains a Gated Recurrent Unit (GRU) [33] with a state size of 20 which finally outputs a 4-dimensional vector after having gone through the sequence of data rows; for further details please refer to Supplementary File 1.
After having encoded the PK data, the model simulation is performed via the "PKODE" submodule, which is a neural network of recurrent architecture that performs the forward Euler time steps [34,7]. Further details of this recurrent architecture are shown in Supplementary Fig. 1(b). As there is no drug concentration in the patients prior to dosing, the network port "InitState" (as shown in Supplementary Fig. 1(a)) is set to a vector of zeros. Dosing enters explicitly in this recurrent network via the network port "Dose". Details on how "Dose" enters into the model is further illustrated in Supplementary Fig. 1(b): the dosed component of the PK state is incremented by the given dosages provided in the data set; note that the nondosed component is not varied via the padding layer [23] "pad 0". The network "PKVF" aims to approximate the vector field of the PK model (see Fig. 1(c)). The layer "Dt*" performs element-wise multiplication by the step size (1/4 day in the current implementation) corresponding to the discretized time rate of change of the PK system. Finally, the rectified linear unit [23] "ReLU" ensures that the PK state remains non-negative. The model construction was implemented in Wolfram Mathematica [35] and is provided in the Supplementary File 1.
Through backpropagation algorithm [23], the trainable weights of "PKEncoder" and "PKODE" sub-modules are iteratively refined to minimize the L2 loss function between the observed data and model outputs. As previously mentioned, we used an 80-20 split of the total patient data. Data augmentation as described in the previous section was performed. Note that once selected, the test patients are never used in the training phase but kept aside for the final evaluation of the network. The ADAM optimizer [23] was used to train the neural network for a total of 2000 epochs. The trained network is then evaluated on the n = 133 test patients, and comparison to the ground truth showed good performance, with r2 = 0.98, correlation coefficient of 0.99 and RMSE=2.67.
Pop-PK/PD model and prediction
We used a pop-PK/PD model with structure as described in [17,18]. In particular, the model consists of a two-compartmental PK model (with 2 state variables and 4 parameters) and the platelet dynamics is described using 6 state variables and 10 parameters that are variable between patients (i.e., where inter-individual variability is allowed) [5]. The model was built sequentially [20,5], with PK parameters first estimated and subsequently followed by the PD parameters using the First Order Conditional Estimation (FOCE) method in NONMEM 7.3.0 [36].
For the pop-PK/PD approach, we process data in the following manner: (a) for patients in the training set, we leave the whole observation and dosing data intact; (b) for patients in the test set, we keep all the dosing data but only retain PK & PD observation data within the initial window t < tObs (i.e., setting "DV" to empty and "MDV"=1 in the NONMEM [36,5] data set).
From this input data, NONMEM [36] was used to carry out estimation of the model parameters both at the population level and the individual level. From the result of the estimation, NONMEM produces amongst its outputs the individual predictions ("IPRED") for platelet dynamics. We make predictions for the unseen portion (i.e., t ³ tObs) for patients in the test set and compare them to the data.
Neural-PD network and training
Having first trained the neural-PK net, we subsequently copied over the trained weights from "PKEncoder" and "VFnet" of Supplementary Fig. 1 onto the corresponding PK components of the neural-PK/PD network shown in Fig. 1. These weights are then frozen from any further training. The n1´5 input data that enters the network port "PKPDData" is then selected for the following 4 columns via the "PDData" sub-module shown in Fig. 1: (1) time-after-dose, (2) time, (3) PK and (4) platelet count; that is, the dosing column is dropped. The implicit assumption being made is that it is the drug concentration which drives the pharmacodynamic effect, but dosing does not directly mediate the pharmacodynamic effect; this is a standard assumption made in PK/PD modeling. The selected data columns are then fed into "PDEncoder", which at its core contains a Gated Recurrent Unit (GRU) [33] with a state size of 50 and outputs a 10-dimensional vector. Finally, both the outputs of "PKEncoder" (4-dimensional vector) and "PDEncoder" (10-dimensional vector) are fed into the "ODE" sub-module, which simulates both the PK and PD components of the model using those encoded vectors, driven by the dosing coming in via the "Dose" network port shown in Fig. 1. In our model, we represent the PD (platelet) dynamics using a 4-dimensional ODE state vector. The underlying assumption that PK drives PD (but not vice versa) is encoded into the network architecture of Fig. 1(b). For the PD dynamics, the corresponding 4dimensional ODE state vector needs to be initialized prior to the first dose being given; this is in contrast to the PK part of the model, where prior to dosing the drug concentrations are identically zero. The combined network (Fig. 1) takes as an input the initial platelet state obtained from looking at the first platelet count after the start of treatment (t ³ 0); this data pre-processing was done for all patients and enters through the network port "InitState".
However, simply taking the platelet estimate prior to treatment as the first observed platelet data is inaccurate as some patients had a marked platelet drop right after the start of treatment; hence we use the "ICNet" to help improve the estimation the initial platelet count.
This was done by estimating a scalar from the early observed data and then replicating it to the 4-dimensional platelet state vector. The need to estimate the initial platelet value from the time course data is similar to that of the pop-PK/PD mylosuppression models [19,17]. The "ICNet" consists of a bi-directional [37] GRU with a state size of 10; see Supplementary File 1 for further details of the implementation. The network port "ObsState" outputs both the PK and PD (platelet count) predictions at each time step. In the sequential training approach, only the PD component is compared to the platelet data, based on the L2 loss function (which computes the mean squared error). The ADAM optimizer [23] was used to train the neural-PK/PD network for a total of 3000 epochs until the training loss no longer improves.
Model Prediction Benchmarking
We ensure that both the pop-PK/PD and neural-PK/PD are compared against the groundtruth platelet data in an identical manner on the test patients, with respect to both r2 and RMSE.
Simulating dosing regimen
While most patients in the training set were given Q3W dosing, a few were given doses at Q1W frequency. Nevertheless, we can use the neural-PK/PD model to simulate the scenario whereby all the patients were given T-DM1 at the dose 3.6 mg/kg Q3W as follows. Firstly, the complete training patient data were fed into the network port "PKPDData" to obtain the encoded parameters outputs from network sub-modules "PKEncoder" and "PDEncoder".
Then, we feed the desired regular dosing of 3.6 mg/kg Q3W into the "Dose" network port.
The 5 th , 50 th and 95 th quantiles of the neural-PK/PD simulated results from the training patients were then computed to generate the plots shown in Fig. 5(a). A similar procedure was used to simulate T-DM1 at the desired dosing regimens in the training patients to generate Fig. 5(b) and 5(c). | 2020-10-23T01:00:49.180Z | 2020-10-22T00:00:00.000 | {
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268161590 | pes2o/s2orc | v3-fos-license | A spatial classification applied to convectional reaction-diffusion boundary problems basing on a geometrical polymorphism of biological objects
Convectional reaction-diffusion is a common physical process which forms a background for existence of all biological objects. Along with the fluid flows it provides a supplying/removing of chemical compounds in a living system. In mathematical modelling PDE border problems are usually used for decryption of such kind phenomena. In the present study the classification of mathematical problems is developed on the base of the general geometrical properties of the considering living objects. Moreover, the uniform/non-uniform and the homogenic/non-homogenic structure of the boundaries and the internal area are taken into consideration. The type of classified models is determined by the structure of both an outer boundary and the inner space of the contemplated objects (i.e. their homogeneity/heterogeneity, H/H Classification). Furthermore, several dimensionless metrics are also introduced to estimate geometrical features influence the spatial-time distributions of chemical compound concentrations. The proposed classification can be applied to any kind of biological systems to consider a spatial polymorphism and its influence on the metabolic gradients.
Introduction
Considering the main properties of biological objects, one obviously highlights that there are several essential features which are inextricably linked to them.First of all, the area of consideration is always 3D.Despite possible symmetry and the difference in the size, it means that the processes must be considered under essential spatial dependence of variables, and, as a result, they are spatially distributed.Furthermore, the distribution could be non-homogeneous and spatially localized in several compartments [1,2].The second aspect of the problem is the mutual combination of different physical and chemical processes [3].The most of them occur simultaneously (figure 1).Under such conditions the complexity of the considered problems seems to be unsolvable, or the ways of simplifications cause distortion of biological formulation background.Another essential aspect of reaction-diffusion with convection is a boundary condition.It is inextricably linked to geometry of the analyzed system and the algorithm of a digital phantom creation.If the distribution of chemical compounds is a subject of the analysis, then biochemical reactions and their spatial non-homogeneity must be taken into account [4].Thus, the final presentation of the biological claims forming a physical model to be modelled is a combination of known important processes.No one of them can be really excluded without losing of essential properties of the considered object.
Figure 1.
A schematic example of a living object with indication of physical processes and structures.A diffusion area is the medium where the chemical compounds (metabolites) can move forming a nonuniform concentration distribution.Convection appears as a result of a solvent movement inside the medium due to a difference of pressure.The area of convection may be an interstitial fluid among cells or especially organized space (not shown) like a blood stream, lymphatic channels, etc.The boundaries are forming the border under where the variables are determined.The medium is filled up with many internal compartments where the biochemical reactions occur.The figure is created with BioRender.com.
Having evaluated different mathematical formulation, one can conclude that the problems of reactiondiffusion with convection are able to be classified by geometrical aspects of the system.The main advantage of such a classification is to get a systemic approach to forming a mathematical formulation of the problem meeting to biological demands and features of the considered object.Indeed, the same mathematical models may correspond to the similar biological systems, but the aim of the modelling causes the final choice.In the present study the classification is represented in terms of the properties of spatial areas and localization of the physical processes.This type of systemic ordering for mathematical problems achieves the maximum connection to biological demands.
Mathematical description of convection reaction-diffusion in a biological system
As per the usual approach, the mathematical formulation of metabolites N diffusion in a limited area Ω can be depicted as follows: and u correspond to metabolites concentration, system parameters and convectional velocity field respectively.The reactions in the medium provided by enzymes activities in the metabolic pathways are described by the term . It also includes the influence of some external parameters such as electric potential and so on.One should note that some indications in equation ( 1) are common but very essential.The shape and the property of a diffusion area ( ) are accompanied with the structure and the type of the boundary conditions for the object border ( ).Usually, both real object geometric parameters mentioned above are represented using the simple primitives.For symmetrical system this way seems to be acceptable.However, it is not always possible because of complexity and asymmetric distribution of geometrical shape of the object.Nevertheless, the geometric classification classes that will be introduced are extremely easy to understand because they are based on the analysis of elementary interactions in set theory.
It is essential to highlight that the introduces classification is quite different from other ones.For instance, the parameter space was earlier fully classified in terms of the types and stability of the uniform steady state for the reaction-diffusion systems of two chemical species on stationary rectangular domains [5].Furthermore, anomalous diffusion in animal movement data can be classified using power spectral analysis [6].Various statistical testing approaches may apply to fractional anomalous diffusion classification [7].Nevertheless, the creation of a new classification is influenced by the requirement to create a phantom basing on real biological geometry.
The general classes of geometrical types of considered problems
As already mentioned above the complexity of a biological system creates formidable difficulties to construct 3D phantom and to set up the boundary conditions in equation (1).Indeed, the most important problem is to choose a type of condition and furthermore evaluate the appropriate value.Nevertheless, all cases can be assigned to two general classes only.Class I represents a ranged area with continual external boundary.The most part of models will get into this class.Indeed, the processes are usually included into the area of consideration as a part of continual properties of the medium.This claim is true for both convection and reaction-diffusion.However, under some conditions one needs to exclude a part of the object area out of scrutiny.Under these circumstances the second Class of the models appears.Its especial feature is a forming of an internal boundary which is the part of the object border, and the boundary conditions are defined there.The schematic illustration of the mentioned classes is represented in figure 2.
Class I Class II
Figure 2. A scheme of two geometrical classes applied to the boundary problem of reaction-diffusion with convection.A green area indicated the diffusion area ( ), and the blue line corresponds to the object border ( ).In both classes the final phantom is formed as a unit of and .
These types of geometry determine the style of the digital phantom creation, and they partially influence the choice of boundary conditions.For example, if a researcher prefers to exclude the part of convection appearing due to a blood flow the area of inner cavity of the blood vessels will be omitted from the phantom.In this case Class I converted into Class II with appropriate boundary conditions on the inner part of .The classification process is further complicated by the properties of and .
The detailed classification of reaction-diffusion problem on the base of homogeneity/heterogeneity of the phantom elements
The properties of the diffusion area and the boundaries are the most essential to be formulated in the body of the digital phantom [8].Modern capabilities allow for the conditions to be considered to be varied.The simplest case is a homogeneous medium with a homogeneous boundary.Under such circumstances the phantom has the following properties: ,; The next variation is characterized by a spatial heterogeneity being present both inside and on its borders.The non-homogeneous conditions in the medium are determined by the conditions: The heterogeneity of the boundary conditions is reflected in possible combination of different type of conditions and\or spatial non-homogeneity of one type of condition. ,; It is essential to notice that classification relies on the combination of multiple specific properties of the diffusion area and its boundaries.For instance, sub-subclass 2 in any Class is caused by the consideration of various phases in the biological object, like the membrane and cytosol, which have different diffusion coefficients.Furthermore, the boundary conditions can be homogenic, but they are combination of the Dirichlet Conditions and the Neumann Conditions on different surfaces.This feature of the problem immediately leads to subclass 2 in H/H Classification.The combination of classes and subclasses is represented in table 1.
. Table 1.The classification of convectional reaction-diffusion problems.The classes and subclasses indicated by the numbers.The main aspect is the combination of homogeneity and heterogeneity for both the diffusion area and the boundaries (H/H Classification).a The classification is represented as the final combination of numbered conditions.For example, Class I with homogenic boundary and homogenic diffusion area will be represented as H/H I.1.1 and for heterogenic diffusion area -H/H I.1.2,etc. b Boundary conditions determined by equations ( 3) and ( 5).c Homogeneity/heterogeneity of the diffusion area according to equations (2) and (4).
The parameters which can be used as metrics to estimate geometrical fluctuation of digital phantoms
Using introduced H/H Classification one is able to formulate clearly the types of the phantom parts and to consider their appropriate properties.However, sometimes the property of the area can be averaged according to geometry and the evaluated values.In particular, each biological surface has roughness, but this may be negligible when a concentration gradient is formed near it.Indeed, this aspect needs to be considered case to case.Nevertheless, some common recommendation of metrics is reasonable to take into account.The considered phantom always has the standard relations between medium properties and the geometrical polymorph sizes.The simplest metric is a relation between a diffusion coefficient and the constant of a medium consumption.If the third term of equation (1) includes the summand like ( ) then the relation is expressed in the following form: This parameter in a modified dimensionless form was firstly introduced for modeling of glucose gradients near pial vessels using sphere sources diffusion fields (SSDF) method [9].However, equation ( 6) may be reasonably modified taking into account the geometric sizes of both the phantom and the roughness.The expression looks like as follows: where roughness is a part of the considered phantom related to irregular geometrical features of the biological object.The value of parameter introduced by equation ( 7) may be a common criterion of geometrical irregularity inclusion/exclusion in creation of a digital phantom.
Discussion
Modelling of reaction-diffusion coupled with convection and the gradients of electric field is always based on precise relation between real shape of the biological object and geometry and parameters distribution in the mimicking digital phantom.Under omitting of some part of geometry or simplifying of physical processes the researcher is in danger of losing significant information about the object being investigated.Therefore, to avoid this risk, one needs to consider right processes with right localization.In particular, for H/H I.2.2 models of glucose distribution in a neurovascular unit it was shown that the geometry of inner flux dysconnectivity barrier essentially determines the reply of the system on changes of cerebral blood flow in the capillary [10,11].The same effect of geometrical polymorphism appears in other system corresponding to H/H I.2.1.It was shown that a spatial polymorphism of the post-synaptic density of glycine receptors is important to form a physiological response to a neuromediator release [12].Thus, the introduced classification can be a useful tool for design of schemes of a digital biological phantom creation and further localization of boundary conditions and internal parameter distributions. | 2024-03-03T19:17:37.088Z | 2024-02-01T00:00:00.000 | {
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259250342 | pes2o/s2orc | v3-fos-license | Young gastroenterologists angle: Friends of the UEG young talent group consensus statement on the structure of young gastroenterology sections
awards, poster prizes, travel grants, etc.). National societies should consider supporting the young gastroenterology section to organize small meetings or workshops with concise topics. Additionally, it is important to establish scientific networks for multicenter studies to involve and empower young gastroenterology researchers. Either, young gastroenterology section research projects can be integrated into existing networks, thereby promoting young researchers in gastroenterology. Alternatively, the YGS can be supported to establish their own network, facilitating collaboration and exchange of ideas between young researchers and institutions. As a future perspective, we see the linkage of several national research networks to a European network of young gastroenterology researchers as a way to foster international multicenter research, and ultimately, improve patient outcomes through increased scientific collaboration. SUPPORT FOR YOUNG SECTIONS IS VITAL TO ENHANCE THEIR IMPACT Besides engagement at national meetings, support by the national society to the young gastroenterology section projects should be provided. Projects could include but are not limited to ‘summer schools’ with a clinical or scientific focus, postgraduate training, or ongoing education possibilities, as well as projects aimed at influencing professional policy on a national level. To meet the needs of the next generation and motivate young volunteers to apply themselves, a high degree of freedom in planning for the YGS is beneficial. Funding and administrative support are vital aspects for the success and growth of young gastroenterology section projects. Each section should establish rules on acceptable sources of funding (e.g. third‐party or industry funding) and conditions for accepting funding from external sources. National gastroenterology societies should communicate a transparent budget for their respective YGS. Administrative support is just as crucial as funding and smoothens cooperation. Therefore, a clerical position in the society's secretariat should support the YGS. THE INVESTMENT IN THE NEXT GENERATION OF GASTROENTEROLOGISTS WILL ENSURE THE SUCCESS OF EUROPEAN GASTROENTEROLOGY In summary, integrating young gastroenterologists into national gastroenterology organizations is essential for the success of national organizations as a whole. Without a clear structure, the long‐term success of YGS hinges on personal enthusiasm and engagement, limiting their positive impact. Any investment in the next generation will bring future dividends for national organizations and the field of gastroenterology. Due to heterogeneous local conditions, the suggestions in this article will need adjustment with local particularities in mind. The involvement of the next generation of gastroenterologists in finding the ideal local structure is crucial. By implementing a clear structure and guidelines, YGS can continue contributing to the growth and success of national gastroenterology associations throughout Europe. Jonas Jaromir Staudacher Johan Burisch Paula Sousa Maciej Salaga Gianluca Pellino NEWS 579
INTRODUCTION
Nevertheless, the structure of young GI sections in UEG member societies is heterogeneous. Some national organizations currently do not have a formal young GI section. 1 Previously, the UEG Young Talent Group published a 'cookbook' on how to start a young GI section 2,3 giving valuable insights into the pivotal steps in the first months. Notably, this document does not sufficiently address the desired long-term structure and maintenance of young GI sections. In our eyes, a durable and practical structure is fundamental to longterm effective cooperation and success. Therefore, we present a consensus on a possible structure for national young GI sections.
TRUE REPRESENTATION OF THE DIVERSE NEXT GENERATION OF GASTROENTEROLOGISTS IS FUNDAMENTAL
A clear definition of who qualifies as a young gastroenterology section member is necessary to accurately represent the next generation. The threshold could be either age (e.g. younger than 40 years) or the state of specialty training (e.g. members who have finished their gastroenterology training in the last 3 years). Exceptions to colleagues with non-linear careers, such as times of childcare or research breaks, are an effective tool to reflect the diversity of individual vitae.
Future gastroenterologists with an academic as well as nonacademic background and a focus on outpatient or inpatient care should be invited to join the young gastroenterology section to manifest the whole breadth of gastroenterology. Successful gastroenterology is diverse and interprofessional, and young GI sections should be open to professionals with a shared interest in gastroenterology besides physicians (e.g. early-career basic and translational scientists, dietologists, and nurse practitioners with a GI or endoscopic focus). Allowing medical students to be part of the Young GI sections or devising specific events targeted at students is another way of raising the interest of potential future colleagues in the field of gastroenterology.
DEMOCRATIC STRUCTURES: INCREASE PARTICIPATION AND TRANSPARENCY
A speaker or spokesperson should be elected by members of the section. To guarantee continuity, a team of speakers and vicespeakers with overlapping terms, and progression from vicespeaker to speaker should be considered. Additional designated posts (e.g. treasurer), should also be determined through election by the section members. Implementing a term limit ensures the ongoing renewal of young gastroenterology section leadership. To promote equality and representation throughout the YGS and to embrace the heterogeneity of the next generation of gastroenterologists, rules on gender, geographic, or ethnic origin of the speaker team (e.g. genderparity with regards to speaker and vice-speaker) should also be considered.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
REGULAR INTERNAL AND JOINT MEETINGS ARE THE CORNERSTONE OF SUCCESSFUL COLLABORATION
Meetings between young section speakers and society representatives are crucial for exchange and maximizing productivity.
Therefore, there should be regular meetings to discuss joint projects, needs, and future directions. At these meetings, the speaker team should represent the young GI section, and a dedicated Here, online or hybrid formats can help to increase participation.
Updates on current and planned projects should be given, and the meetings should serve as a forum for feedback from and involvement of all young gastroenterology section members. A culture of transparency helps to improve participation and foster a feeling of belonging.
THE CONSIDERABLE EXPERTISE HELD BY THE NEXT GENERATION OF GASTROENTEROLOGISTS IS A RESOURCE THAT SHOULD BE UTILIZED
The expertise held by members of the next generation should be utilized in national meetings, including (co-)chairing sessions and giving lectures. The YGS can help identify suitable members for any specific theme. A quota of (co-)chairs from the next generation at meetings is a possibility to ensure adequate representation. Additionally, opportunities to independently plan sessions should be implemented so that the focus of the next generation has the forum it deserves. Also, YGS should be involved in choosing recipients for awards, particularly awards promoting the career development of the next generation (e.g. abstract awards, poster prizes, travel grants, etc.). National societies should consider supporting the young gastroenterology section to organize small meetings or workshops with concise topics.
Additionally, it is important to establish scientific networks for multicenter studies to involve and empower young gastroenterology researchers. Either, young gastroenterology section research projects can be integrated into existing networks, thereby promoting young researchers in gastroenterology. Alternatively, the YGS can be supported to establish their own network, facilitating collaboration and exchange of ideas between young researchers and institutions.
As a future perspective, we see the linkage of several national research networks to a European network of young gastroenterology researchers as a way to foster international multicenter research, and ultimately, improve patient outcomes through increased scientific collaboration.
THE INVESTMENT IN THE NEXT GENERATION OF GASTROENTEROLOGISTS WILL ENSURE THE SUCCESS OF EUROPEAN GASTROENTEROLOGY
In summary, integrating young gastroenterologists into national gastroenterology organizations is essential for the success of national organizations as a whole. Without a clear structure, the long-term success of YGS hinges on personal enthusiasm and engagement, limiting their positive impact. Any investment in the next generation will bring future dividends for national organizations and the field of gastroenterology. 5 Due to heterogeneous local conditions, the suggestions in this article will need adjustment with local particularities in mind. The involvement of the next generation of gastroenterologists in finding the ideal local structure is crucial. | 2023-06-27T06:17:03.610Z | 2023-06-25T00:00:00.000 | {
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113654764 | pes2o/s2orc | v3-fos-license | Mechanical behavior analysis of a submerged fixed point anchoring system for a hydroacoustic signature measuring sensor for divers and ships
The paper has as its main objectives the presentation and the analysis of the numerical analysis results for the study of a fixed point anchoring system for a hydroacoustic sensor when measuring the hydroacoustic signature of divers and ships in real sea conditions. The study of the mechanical behavior of this system has as main objectives the optimization of the shape and weight of the anchorage ballast for the metallic structure while considering the necessity to maintain the sensor in a fixed point and the analysis of the sensor movements and the influences on the measurements caused by the sea current streams. The study was focused on the 3D model of metallic structure design; numerical modeling of the water flow around the sensor anchoring structure using volume of fluid analysis and the analysis of the forces and displacements using FEM when needed for the study. In this paper we have used data for the sea motion dynamics and in particular the velocity of the sea current streams as determined by experimental measurements that have been conducted for the western area of the Black Sea.
Introduction
Diver and ship hydroacoustic signature is a high importance data which must be known for recognition. For this reason there is a need to create a database for these types of acoustic signatures. To measure these hydroacoustic signatures, it is needed to mount some sensors on the bottom of the sea. These sensors are mounted and pointed in fixed positions for measuring and must be kept in position, within some acceptable limits, until the measuring is finished. The sensor can be kept in fixed position using an anchoring system and appropriate anchorage ballast for it. This study was focused on the volume of fluid (VOF) and FEM models [1,2] optimization of a 3D model of a metallic structure design and its anchorage ballast. As mentioned, our two main objectives for the study were to analyze the mechanical behavior of the anchoring system and to find a proper shape and weight for the anchorage ballast while taking into account the movement limits for the measuring sensor. To achieve these required goals, through the study, we considered some working hypothesis for the simplification of numerical model, such as: the bottom of the sea is perfectly horizontal, we study the fluid structure interaction (FSI) for the sea current streams by studying separately the flow around the ballast and around the metallic structure under the influence of the sea current streams at the measuring depth, we coupled the system of acting hydrodynamic forces on the metallic structure and [3,4], when modeling the water flow around the metallic structure or around the ballast we have taken into account only the velocity of the sea current streams. The 3D metallic structure shape is a triangular pyramid, is presented in Figure 1 and was designed using Solidworks. Cylindrical and conical ballast 3D models are presented in Figure 2 and Figure 3. metallic structure modeling for sea current streams with speeds of 1 m/s, 1.2 m/s, 1.5 m/s and 2 m/s and assessment of the forces applied on it, 3D FEM simulation for displacements and forces in structure evaluation using 3D flow data and body characteristics for the metallic structure with ballast mounted.
Initial data and the study diagram
The study of the mechanical behavior of this system has as main objectives the optimization of the shape and weight of the metallic structure anchorage ballast while considering the necessity to keep the sensor in a fixed point and the analysis of the influence of sea current streams and sensor movements on the measurements.
To accomplish these objectives we conducted sixteen simulation scenarios for the water flow around the ballast for the cylindrical shape and the conical shape. These simulation scenarios used as variable parameters the water flow velocity and the inclination angle of the ballast cylinder shape generator and the height of this one. Figure 4 presents the parametric 3D shapes used for simulation and Table 1 presents the geometrical characteristics of the shape. The metallic structure was designed as a 3D model and is presented in Figure 5. The flow around the metallic structure was analyzed for sea current streams velocities of: 1m/s, 1.2 m/s, 1.5 m/s and 2 m/s. The data obtained from the flow around the metallic structure is used as input data for the 3D FEM analysis in which forces and displacements were numerically calculated for the structure. The schematic of the study organization and data flow between the studied problems is presented in Figure 6. As can be seen in Figure 6 the study was conducted in four main steps: 3D modeling design, 3D flow simulation using the VOF method and the proper turbulence models [3,4], forces and torques numerical determination for ballast configurations considered and the acting pressure on the metallic structure, 3D FEM simulation to evaluate the efforts and displacements in the sensor anchoring structure.
In the following section we present the results obtained for every step mentioned.
Numerical results
The numerical results obtained by simulations are presented as follows and the order of presentation is given by the study diagram presented in section 2, Figure 6.
Sea water flow simulation results around the ballast
The water flow simulation around the ballast was designed on four ballast configurations, for sea current streams with velocities of 1m/s, 1.2m/s, 1.5m/s, 2 m/s. The purpose of the 3D flow simulation around the ballast was to calculate the forces and moments for the center of gravity of each ballast configuration and choose the most appropriate from these for usage in the anchoring system configuration on the bottom of the sea when measuring divers and ships hydroacoustic signatures.
This shape of ballast chosen is presented in Figure 2, Figure 3 and Figure 4 and represents a configuration easy to make using simple and low cost machining techniques.
Once the forces and moments have been determined we decided which of the ballast bodies to use with the metallic structure to keep the measuring sensor in a fixed point on the bottom of the sea.
In Table 2, Table 3, Table 4 and Table 5 are presented the results obtained for torques and forces obtained for each ballast body configuration depending of the water flow scenario analyzed. Table 2 is not an acceptable solution. The total force acting on the ballast body in case of a sea current stream with a velocity of 2 m/s represents 24.20 % of the total weight of the ballast which is not a solution for our sensor anchoring system. To this value we add the weight of the metallic structure and the weight of the ballast body. At the same time the total torque obtained is very high for the sensor and also creates a large displacement which can alter the measuring results for the hydroacoustic signature. The theta angle parameter of 90 degree generates a cylindrical shape for the ballast body, configuration I which is not that good from the point of the water flow characteristic and trajectory. In addition, the total mass of 25.62 kg is high when considering the total mass of the anchoring structure.
The results of Configuration II are presented in Table 3. From these we can see that the total force acting on the ballast in case of a sea current stream with a velocity of 2 m/s represents 22.83 % from the total weight of the ballast which is not a solution for our sensor anchoring system.
The total weight of the ballast for this configuration is 23.48 kg and is high. Total force which acting on this configuration is reduced with 7.21 N as against configuration I and total mass is reduced with 2.14 kg. As we can observe if we reduced the angle theta with 1.66 degree we can obtain a mass reduced with 2.14 kg in conditions of a material defined (aluminum alloy). Configuration's III results are presented in Table 4. Total force obtained in this case is 50.935 N. Total force in this case is reduced with 9.892 N than configuration I. Total force in case of a sea stream velocity of 2 m/s represents 22.48 % from the total weight of the ballast which is still not a solution for our sensor anchoring system.
Configuration's IV results are presented in Table 5. The total force obtained for this configuration is 46.612 N and total mass 19.53 kg. These are lower than the other three configurations. The difference in total force for this configuration compared with the first configuration is 13.215 N. Also we can observe that once we reduce the generator line angle, the mass of the ballast and the force acting on it decrease. Less force and less weight represent the solution we looked for and we considered this configuration of ballast to be the one needed for our anchoring system. For this configuration of ballast we have represented in Figure 7 and Figure 10 the flow trajectories and the pressure flow field. The flow around the ballast body is characterized by low turbulence; the differences in pressure are not so high from one stream velocity to another (Figure 9 and Figure 10). In the scenario with a sea current stream velocity of 2 m/s we can observe much more detachments of the flow around the ballast body and behind it (see Figure 7 and Figure 8). We also have a stream velocity increased by almost 50% from the free sea current stream velocity in both scenarios.
Sea water flow simulation results around the metallic structure
The simulation of the water flow around the metallic structure was made with the purpose to obtain data about the mechanical strains acting on the structure and which are caused by the sea current streams. The results obtained after the simulation of the mechanical strain on the metallic structure are presented in Figure 11 for the sea current stream velocity of 1 m/s and Figure 12 for the sea current stream velocity of 2 m/s. The flow around the metallic structure is not characterized by high turbulence and the mechanical strains on the metallic structure are low. Figure 11. Metallic structure mechanical strain distribution caused by the sea current stream flow with 1 m/s velocity As we can see in Figure 11 and Figure 12 the influence of the flow is not large. The mechanical strains of the water flow on the metallic structure were loaded in the FEM simulation for force and stress evaluation on the anchoring structure.
3D FEM simulation results
The FEM simulation is made on the metallic structure presented in Figure 5. The external loads used are: the weight of the structure, the weight of the ballast, the forces and torques acting on the ballast, the strain forces acting on the structure. As fixture the system was considered fixed in three points. The simulation was made for two scenarios: sea current streams of 1 m/s velocity and sea current streams of 2m/s velocity. The results for the stress (von Mises) are presented in Figure 13 and Figure 14. Total displacements, and axes displacements are presented in Figure 15 and Figure 16.
Conclusions
This paper had as its main objectives the analysis of the numerical results concerning the study of an anchoring system for a measuring sensor in a fixed point for measuring the hydroacoustic signature for divers and ships in real sea conditions. The study of this system's mechanical behavior had as main objectives the shape and weight optimization for the anchorage ballast of the metallic structure while considering the necessity to keep the sensor in a fixed point and the analysis of the sensor displacements influences on the measurements caused by the sea current streams. The study gave us the opportunity to choose from four different configurations of different shapes and weight for the ballast body. The variant proposed in configuration IV has been chosen, with a weight of 19.53 kg and the main geometrical characteristics presented in Table 1. For this ballast body configuration we have evaluated the forces and displacements in the anchoring structure caused by sea current streams of 1 m/s velocity and 2 m/s velocity. After the FEM analysis, we have found a maximum stress of 13.822 MPa which is less than 50% of the maximum acceptable stress. The maximum value of displacements was of 1.032 mm. This value of displacement does not have influences on the measurement sensor when measuring at a fixed point. In the end we think that the study achieved the goals required for it. As a future work, there is a need to determine the optimum material for the construction of the ballast body and for the metallic structure and also to study more carefully the hydroacoustic sensor directivity parameters diagram while considering at the same time the displacements found in the anchoring structure.
Acknowledgement
This paper has been financially supported within the project titled "System for detection, localization, tracking and identification of risk factors addressing importance strategic objectives in littoral areas", contract number 302 cod PN-II-PT-PCCA-2013-4-0377. This project is financed by PNII. | 2019-04-15T13:06:18.872Z | 2016-08-01T00:00:00.000 | {
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220880729 | pes2o/s2orc | v3-fos-license | Multimodal therapy with surgery and adjuvant nivolumab for late-onset multiple liver metastases of choroidal malignant melanoma: a case report
Background Choroidal malignant melanoma is the most common primary malignant tumor of the eye in adults. Prognosis after recurrence of this disease has been dismal because of the absence of an effective therapy. However, resection of recurrent foci and a subsequent treatment with immune-checkpoint inhibitor may improve the prognosis after recurrence of this disease. This study presents a case of late-onset liver metastases of choroidal malignant melanoma, successfully treated with hepatectomy and postoperative adjuvant nivolumab. Case presentation A 53-year-old woman had undergone left ocular enucleation because of choroidal malignant melanoma 13 years prior to admission. She visited a nearby clinic with complaints of epigastric pain. She was referred to our hospital because a giant liver tumor was observed on abdominal ultrasonography. Enhanced computed tomography revealed multiple liver tumors in the right lobe, 49 mm in diameter with ring enhancement in subsegment (S) 5/6, and 14 and 8 mm without any enhancement in S7 and S5, respectively. On magnetic resonance imaging, the main tumor showed high intensity on T1-weighted with fat suppression, suggesting melanin deposition. Based on the diagnosis of multiple liver metastases of choroidal malignant melanoma, right hepatectomy and regional lymphadenectomy were performed. She was discharged without postoperative complications. Histological examination revealed that all tumors were metastatic malignant melanoma. She was treated with nivolumab postoperatively, and no recurrences were observed during 22 months of follow-up. Conclusions Aggressive surgery plus adjuvant nivolumab appears to be a promising treatment for choroidal malignant melanoma with late-onset liver metastases.
Background
Choroidal malignant melanoma is the most common primary malignant tumor of the eye in adults. It is highly likely that micro-metastases of malignant melanoma originating from the eye via systemic circulation have previously occurred in other organs following the detection of liver metastases of this disease. Therefore, local therapy, including liver resection, is not generally acceptable as a curative therapeutic option [1]. Although patients may benefit from liver resection in selected cases, patients with metastatic melanoma have a median survival of 6 months [2][3][4].
Currently, immunotherapy is the standard therapy for unresectable or metastatic melanoma, and the prognosis of metastatic melanoma has improved [5]. Therefore, resection of recurrent foci without morphological residual lesion plus adjuvant immunotherapy with immunecheckpoint inhibitor may be beneficial.
This study reports a case of late-onset liver metastases of choroidal malignant melanoma, successfully treated with hepatectomy and postoperative nivolumab.
Case presentation
A 53-year-old woman underwent left ocular enucleation for choroidal malignant melanoma 13 years prior to admission. Histopathological examination of the enucleated left eye showed a spindle-type choroidal melanoma that did not invade the sclera. Subsequently, the patient was followed up annually by an ophthalmologist (YH).
She visited a clinic with complaints of epigastric pain. A large liver tumor was observed on abdominal ultrasonography, and she was referred to our hospital. There were no elevations in serum tumor markers (AFP, PIVKA-II, 5-SCD, or NSE), and her liver was in good condition: Child-Pugh score was 5 points, class A. Liver damage was defined as grade A, according to the Liver Cancer Study Group of Japan [6]. Endoscopy showed no gastrointestinal malignant lesions. Enhanced computed tomography (CT) revealed multiple liver tumors in the right lobe, 49 mm in diameter with ring enhancement in subsegment (S) 5/6, and 14 and 8 mm without any enhancement in S7 and S5, respectively (Fig. 1). Lymph node swelling was not observed. Lung CT revealed no definite lung metastasis except for a small nodule, which had already been detected during the primary surgery. On magnetic resonance imaging (MRI), the main lesion showed very high intensity on T1-weighted with fat suppression, suggesting melanin deposition (Fig. 2). The other two lesions indicated low and high intensities on T1-weighted with fat suppression and on T2-weighted imaging, respectively. Subtraction images displayed enhancement in all three tumors. Whole-body F-18-fluoro- Fig. 1 Enhanced CT (computed tomography) findings. a Enhanced CT showed a tumor with ring enhancement in S5/6 (black arrow). c, e S5 and S7 tumors were not observed in the early phase (black arrowhead, dotted black arrow). b, d, f All three tumors were visualized as low-density regions in the late phase 2-deoxyglucose (FDG) positron emission tomographycomputed tomography (PET-CT) scan revealed FDG deposition in the main tumor, unlike the others (Fig. 3). These tumors were diagnosed as multiple liver metastases of choroidal malignant melanoma.
A right hepatectomy was performed. Black swollen lymph nodes in the hepatoduodenal ligament were resected based on the suspicion of lymph node metastases (Fig. 4). The operation time was 262 min, and blood loss was 380 ml. She was discharged without postoperative complications.
On gross inspection, the S5/6 and S5 tumors were observed to be melanotic, unlike the third tumor (Fig. 5). On microscopy, spindle-shape atypical cells were Fig. 2 Gadoxetic acid-enhanced MRI (magnetic resonance imaging) findings. a-d S5/6 tumor showed very high-intensity regions on fat suppression T1-weighted imaging (fsT1WI), low intensity on fsT2WI, high intensity in dynamic Ph1, and strong enhancement in subtraction images (dynamic Ph1 -fsT1WI) (white arrow). e-l S5 and S7 tumors were visualized as low-intensity regions on fsT1WI, high intensity on fsT2WI, iso-intensity in dynamic Ph1, and weak enhancement in subtraction images (dynamic Ph1 -fsT1WI) (white arrowhead and dotted white arrow) observed in all three tumors. The S5/6 tumor showed strong immunostaining for Melan A, HMB45, and S-100, while the other two tumors showed weak staining (Fig. 6). Programmed death (PD)-ligand 1 expression was not evaluated. Although immunoreactivity was partially different among the three tumors, all tumors were diagnosed as malignant liver metastases with spindleshape atypical cells and HMB-45-positive cells. Spindleshape atypical cells were not observed in the perihilar black lymph nodes, although there was a rich melanin deposition. Genetic screening revealed the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) status as wild-type. She was treated with nivolumab (3 mg/kg) every 2 weeks, and she was followed up with whole-body PET-CT and head MRI scans every 6 months according to the National Comprehensive Cancer Network guidelines for cutaneous melanoma [7]. No recurrence was observed for 22 months after surgery. She has not suffered from any serious adverse events.
Discussion
Choroidal malignant melanoma is the most common primary malignant tumor of the eye in adults, although there are only about 50 cases per year in Japan [8]. Despite local optimal treatment (surgery or radiation) for primary lesion, choroidal malignant melanoma occasionally shows late-onset metastasis: the median disease-free interval is 22.3 years [9]. The prognosis after detection of late-onset recurrence of choroidal malignant melanoma has been dismal. Because of high blood flow to the choroid, the mode of metastatic spread of choroidal melanoma is supposedly hematogenous, with the liver being the most common site [10]. For a long time, liver resection of melanoma had remained controversial. Despite attempts at curative resection, the overwhelming majority (75%) of patients with metastatic melanoma experienced disease recurrence [11]. The median time to recurrence after hepatic resection was 8.3 months. Mariani et al. reported on the surgical management of liver metastases from uveal melanoma [12]; four important survival indicators were described as follows: long disease-free interval (> 24 months), small number of recurrent foci (≤ 4 lesions), comprehensiveness of resected metastases, and the presence or absence of miliary disease. Similarly, Herman et al. suggested the following criteria suitable for resection: disease-free interval > 24 months, absence of extra-hepatic disease, presumed completely resectable lesions, and absence of clinical comorbidities [2]. This case met all aforementioned criteria. A recent meta-analysis still indicated that radical resection of liver metastases from melanoma improves the overall survival compared with non-operative management or incomplete resection [13]. For this reason, radical liver resection was primarily selected.
Nonetheless, adjuvant chemotherapy has been used to extend survival. Patients receiving some form of systemic therapy had longer median survival than patients who exclusively underwent surgery [11]. Nevertheless, the optimal chemotherapy regimen has not been determined.
Nivolumab, a human IgG4 monoclonal antibody against PD-1, and ipilimumab, a human IgG1 monoclonal antibody against cytotoxic T-lymphocyte antigen 4, are approved in several countries for the treatment of metastatic melanoma [5,14,15]. There is an adjuvant treatment strategy in patients with sufficiently high risk of developing metastatic disease. Recently, the Food and Drug Administration approved nivolumab as adjuvant therapy in patients with resected stage IIIB, IIIC, or IV melanoma [5]. Patients who received nivolumab as adjuvant therapy after tumor resection benefitted regardless of the PD-L1 or BRAF status. Under treatment with nivolumab, the estimated 1-year recurrence-free survival in patients with stage IV is 63% [5]. Although the result of overall survival and efficacy for organ metastasis remain unclear, aggressive liver resection can be performed even in the context of multiple liver metastases because there is supporting evidence for the use of adjuvant chemotherapy (nivolumab). Conversion surgery after nivolumab is one of the treatment options to avoid the possibility of an early recurrence. On the contrary, PD-1 antibody is supposedly not effective for choroidal malignant melanoma, unlike cutaneous malignant melanoma [16]. It is reasonable and technically feasible to primarily select surgery in the case of optimal liver function. Combination therapy of nivolumab and ipilimumab for metastatic uveal melanoma appeared to be significantly superior to monotherapy [17]. However, 39.1% of patients treated with combination therapy experienced severe adverse event. Fig. 6 Histopathological examination of the tumors. a, e, i Spindle-shape atypical cells were seen in all three tumors in HE staining. b, c, d The S5/6 tumor showed strong immunostaining intensity for Melan A, HMB45, and S-100, respectively. f-h S5 tumor showed weak immunostaining intensity for Melan A, HMB45, and S-100, respectively. j-l S7 tumor showed very weak immunostaining intensity for Melan A, HMB45, and S-100, respectively. m-p Tumor cells were not observed in the lymph nodes, although the melanin was very rich (white arrow) Based on the results of phase 3 trial, it is considered that the adjuvant chemotherapy (nivolumab) may be terminated if there is no recurrence 1 year after surgery [5]. In this case, we similarly planned to terminate nivolumab because no recurrence was observed 1 year after liver resection. However, a preoperative CT scan revealed a small nodule in the lung. This nodule had already been detected at the time of the primary surgery (13 years before) and was not changed when liver metastases were detected. Prior to the hepatectomy, we consulted with a respiratory surgeon and opted for observation unless the pulmonary nodule would become enlarged. The pulmonary nodule has been unchanged until the most recent PET-CT scan. Although we continue nivolumab following the patient's explicit request, no remarkable changes were observed 22 months after the liver resection.
Patients with amelanotic metastatic melanomas had longer survival time than those with pigmented tumors [18]. Tumor appearance depends on the melanin volume, and the pigmentation level reflects malignant potential. In this case, macroscopic examination indicated that two tumors in the anterior section were melanotic, unlike the third tumor, which was not. We could not find any reports supporting this unique event. Notably, immunostaining for HMB45 is useful in differentiating malignant melanoma from benign, even if lesions are amelanotic [19].
Importantly, in radiologic imaging, tumor appearance can similarly depend on the melanin volume. Gadoxetic acid (Gd-EOB)-enhanced MRI is more useful than multidetector CT for the detection of multiple metastatic foci [20]. It can provide information not only about the vascularization of the lesions in different phases of contrast circulation but also the functional parameters in the delayed hepatobiliary phase. However, it is difficult to value the vascularity because of high-signal intensity in pre-contrast T1-weighted imaging [21]. In this regard, subtraction technique should be used to evaluate enhancement.
In this case, perihilar black lymph nodes were resected under the suspicion of lymph node metastases; however, these nodes pathologically showed no metastatic melanoma cells. Assumingly, either the perihilar lymph nodes took up the melanin released from hepatic metastases or the metastatic melanoma cells were degraded by immunocytes, and only melanin could be deposited in the lymph nodes; however, the evidence is lacking.
Conclusions
Multimodal therapy with surgery and adjuvant nivolumab has the potential of improving the prognosis of late-onset liver metastasis from choroidal melanoma. | 2020-07-31T14:57:38.999Z | 2020-07-31T00:00:00.000 | {
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269939749 | pes2o/s2orc | v3-fos-license | Study on Working Characteristics of 4-Column Hydraulic Support in Lifting–Lowering–Moving State Based on Microcontact Theory and Rigid–Flexible–Mechanical–Hydraulic Coupling Simulation Model
: A hydraulic support is one of the most important pieces of equipment in fully mechanized coal mining, and its stability and reliability will have a direct impact on fully mechanized coal mining. In order to deeply elucidate the dynamic working characteristics of a hydraulic support during lifting, lowering, and moving, and to provide theoretical support for further optimizing the stability and reliability of a hydraulic support, the dynamic characteristics of a hydraulic support are studied in this paper. Firstly, in order to study the dynamic working characteristics of hydraulic support lifting, a rigid–flexible coupling dynamic simulation model of a hydraulic support is established; in order to study the dynamic working characteristics of hydraulic support moving, a microcontact dynamic model of a hydraulic support and the caving face roof and floor based on G-W contact theory is proposed, and the first rigid–flexible–mechanical–hydraulic coupling dynamic simulation system of a hydraulic support and the roof and floor of a caving face is established in the industry. Then, based on this foundation, simulation experiments are conducted for hydraulic support lifting, moving without pressure, and moving with pressure, respectively. The working characteristic parameters of the hydraulic support are collected and analyzed. The results show that working speed, working height, surface contact conditions, residual working resistance, and impact load have different effects on the stability and reliability of the hydraulic support. This study can provide in-depth technical support and theoretical guidance for understanding and improving the dynamic working characteristics of the hydraulic support.
Introduction
Coal is China's most important primary energy source.Up to 2050, coal will still make up over 54 per cent of Chinese energy consumption.Therefore, in the near and long term, the safety, efficiency, and cleanliness of coal resources will remain a significant problematic for Chinese coal industry [1][2][3].Intelligent mining is the key link to realize the intelligentization of coal mines, and it is also the core technical means to reduce the number of caving faces and ensure the safety of personnel [4][5][6].The automated continuous and stable coordinated operation of a set of fully mechanized mining equipment is the foundation of smart mining [7,8].A hydraulic support provides a safe working space for caving face and promotes its operation through interaction with surrounding rock and mining equipment.It is one of the most significant pieces of equipment in the fully mechanized coal mining face [9].In addition to bearing static gravity load from the roof, the hydraulic support also needs to cooperate with a shearer and scraper conveyor to perform Model.Actuators 2024, 13, 193.h ps://doi.org/10.3390/act13050193dynamic movement of lowering, moving, and lifting to achieve the purpose of controlling the roof.The hydraulic support's working performance has a significant impact on the stability and reliability of coal caving face mining [10].Therefore, carrying out in-depth research on hydraulic supports holds considerable significance.
Many scholars have studied hydraulic supports.With respect to the response and load characteristics of a hydraulic support, Zeng et al. discussed the different responses of a hydraulic support in the process of roof rotation by employing a mechanical-hydraulic joint simulation model.Based on this model, the working law of a hydraulic support is analyzed, and the results show that the peak values of pressure and flow increase continuously with the increase in roof rotation speed [11].Ren et al. designed a hydraulic support model with a reduced scale of 1:2, analyzed its response characteristics under a dynamic impact load and verified the dynamic impact experiment of the whole hydraulic support in ADAMS.The experimental results show that the impact resistance of a hydraulic support depends largely on the initial support conditions, and different vertical stiffness will affect the energy distribution ratio of the whole support system [12].Zeng et al. studied the influence of pin clearance on the a itude and dynamic characteristics of a hydraulic support considering the different distribution of pin clearance.The results show that when the front end of top beam is loaded, the mechanical curve of each hinge point is higher than that of the rear end.The maximum load fluctuation coefficient reaches 1.04 for the hinge point on the side without clearance, while for the hinge point on the side with clearance, some hinge points cannot play a supporting role [13].Wang et al. studied the adaptability of impact dynamic load under roof beam on the basis of analyzing specific parameters.In addition, the impact coefficient I and excitation coefficient E are introduced to transform the analysis results, and the evaluation method of response degree of top beam under the impact load is optimized, and the adaptability variation law of impact dynamic load under different impact working conditions is explored [14].Yang et al. simulated the stress state of the hinge joint of the support when different forms of loads acted on the roof beam, and identified the critical loads of different hinge joints.The results show that under the impact load of 6000 kN, the hinge joint between the roof beam and the shield beam is the most dangerous hinge position [15].Xie et al. studied the force response characteristics of the hinge joint between the roof beam and the shield beam and the vibration response characteristics of the column system when the roof beam and the shield beam are subjected to impact loads.The results show that the dynamic response of each hinge point of a hydraulic support reaches peak value when the impact load acts on the roof beam and shield beam simultaneously.As the impact load moves toward the goaf, the hinge point force presents different pressure relief characteristics [16].Zeng et al. studied the dynamic response characteristics of the shield support supported by four pillars under symmetrical and asymmetrical load conditions when the roof beam and shield beam bear the impact load.The results show that under the conditions of a single roof beam impact load, the front pillar is most sensitive to the impact load at the front end of the roof beam.When considering different initial load ratios of columns, the hinge point of the top roof-shield beam has the highest sensitivity to the change in initial load ratio, and when the initial support force of front column is insufficient, the bearing capacity of the support will be weakened the most [17].Meng et al. studied the load distribution law of a hydraulic support bearing points under an impact load.The results show that different hinge points show typical non-uniformity characteristics in the loading process, and the proposed mechanical-hydraulic coupling collaborative simulation method can more accurately obtain the hinge risk points of a hydraulic support [18].Meng et al. established a dynamic simulation model of a hydraulic support based on rigid-flexible coupling, and obtained the force transmission law of support nodes by applying impact loads to different positions on the top beam.The results show that under static load, the support presents obvious variable stiffness characteristics, and the load borne by the rear end of the top beam is higher than that of the front end [19].Cao et al. applied impact loads to different positions of the front roof beam of the support in order to study the stress variation and adaptability of the ultra-high hydraulic support under impact loads.By doing so, dynamic response characteristics of columns and hinge points at different positions and strengths can be obtained.The research results show that when impact loads of different intensities act on the whole front roof beam of the support, the force borne by the pin shaft at the column and hinge point will produce a greater impact, thus reducing the adaptability of the support [20].
With regard to the position and a itude analysis of a hydraulic support, Zeng et al. proved the necessity of studying the clearance of a hydraulic support and analyzed the influence of the clearance between front and rear columns, the clearance size, and the different oil inlet driving modes on the stability of a hydraulic support, and then considered the a itude change in a hydraulic support caused by clearance and clearance size [21].Ge et al. proposed a virtual adjustment method of the support a itude under the propulsion state of a hydraulic support group, and tested it in the Unity 3D virtual software package [22].Hao et al. used virtual and physical point clouds to reconstruct the a itude of the shearer and hydraulic support group in turn to evaluate the risk level of reconstructed a itude of the hydraulic support group, and proposed a method to evaluate the a itude risk level of the hydraulic support group based on reconstructed a itude information.Experimental results confirmed that the method based on point cloud and digital twin is feasible in the a itude reconstruction of the hydraulic support group [23].Shi et al.'s analysis of factors affecting operation performance by hybrid machine learning model for operation state provides eight indexes for evaluating a backfilling hydraulic support, and the results show that the relevant evaluation results are in good agreement with the actual support interval of a backfilling hydraulic support [24].Guo et al. designed a miniature four-pillar hydraulic support for caving coal and performed a non-standard design transformation on the components needed for the hydraulic system.Based on an RC model concept, a perfect hydraulic measurement and control system was configured, and the mechanical properties of the support were tested accordingly.The results show that the micro-support can meet the controllability and multivariate requirements in the test by controlling the pump pressure and cooperating with the hydraulic valve under the premise of similar structure and bearing characteristics [25].Liu et al. established 30 numerical simulation models for experiments by using a discrete element method numerical simulation software package based on a continuous medium.The results show that with the increase in the caving interval, the amount of single caving increases [26].Jiao et al. reconstructed the position and a itude of the hydraulic support, completed the virtual decision making, virtual execution, and strategy optimization of the virtual space adjustment behavior, and finally realized the self-adjustment of the position and a itude of the support [27].Li et al. realized different parameter configurations in the process of following machine automation according to the principle of three-machine cooperation, an established support behavior process decision model, and a supporting equipment linearity collaborative control model of coal mining technology, and previewed the decision model in a virtual scene.Finally, the TCP protocol was used to establish virtual and real interaction channels, and the decision model was verified via a semi-physical simulation experiment [28].Based on the stable pressure supply principle of adapting a reasonable liquid supply flow rate in advance according to the action characteristics of the support, combined with the characteristics of a multi-pump + variable-frequency liquid supply mode, Fu et al. put forward the overlapping cooperative control logic of liquid supply and support; according to the principle of hydraulic transmission, they derived the solution equation of a hydraulic support by following the speed and hydraulic system pressure change rate under overlapping cooperative logic [29].Cai et al. proposed a relative a itude reconstruction method of a hydraulic support based on a digital twin drive.The accurate position and a itude of the hydraulic support were deduced in the virtual space driven by the actual laser sensor information, and the 3D a itude reconstruction was realized [30].
On the subject of hydraulic support structure optimization, Hu et al. studied the discriminant formula of a mobile ram participating in the mechanical balance of the support and the stability judgment criterion of the standard form of the support critical load.The results show that the mobile ram participates in the mechanical balance of an up-down inclined working face, which greatly improves the stability of the support [31].Wan et al. proposed a new balance jack structure and verified the performance of the pressure relief buffer protection process based on AMESim, optimizing the discharge hole.The results show that this new type of balance jack can realize rapid buffer unloading when the hydraulic support is impacted, and has be er impact resistance [32].Stoiński et al. performed bench tests of hydraulic cylinders with a diameter of 0.32 m to verify the calculations and test methods.The results indicate that the best and economically sound direction is to use numerical analysis for model testing and validate these results based on studies of hydraulic leg models used at reduced scales [33].Zhang et al. analyzed the mechanical properties of hydraulic supports under full impact and partial impact of the roof, designed the anti-impact structure, and proved the reliability of the structure.The results show that the anti-impact structure reduces the stress and stress fluctuation of the column, and effectively reduces the deformation of the weak structure of the hydraulic support [34].Zeng et al. established a two-way FSI model based on FSI theory, and analyzed the structural change in the column and the flow field characteristics in a cylinder block under an impact load.The results show that the impact load has a significant impact on the hydraulic cylinder, the cylinder pressure increases, and eddy currents appear on both sides of the bo om [35].
The above research focuses on many aspects of hydraulic supports, such as load-bearing performance, response characteristics, position analysis, structural optimization, and response analysis.In the fully mechanized caving face, the hydraulic support not only withstands the static load from the roof but also collaborates with the shearer and scraper conveyor to dynamically lower, move, and lift, thus facilitating roof control.It is imperative to study the working characteristics of a hydraulic support in this dynamic process, but the existing research in this field is limited, and there are many aspects to be improved.Firstly, the load of a hydraulic support is equivalent to one or more forces in previous studies, but the actual situation should be the complex contact force between a hydraulic support and the caving face roof and floor, which will lead to inaccurate analysis results.Secondly, the research on hydraulic support moving is experimental, and the main focus is on analyzing and optimizing the hydraulic system, but there is limited clarity regarding the working response characteristics of the hydraulic support itself.Thirdly, previous studies focused on the different ways and methods of the support moving with pressure, and did not study the difference in working characteristics between the support moving with pressure and the support moving without pressure.Finally, existing studies have analyzed the lifting or moving process separately, without systematically studying it as a whole.
For the purposes of further clarifying the dynamic characteristics of the support lifting, lowering, and moving, a microcontact dynamic model of a hydraulic support and the caving face roof and floor based on G-W contact theory is proposed, and a rigid-flexiblemechanical-hydraulic coupling model of a hydraulic support and caving face is established.This paper provides the parameters for contact simulation between a hydraulic support and the caving face roof and floor, an accurate simulation model for a hydraulic support moving, and in-depth technical support and theoretical guidance for understanding and improving the dynamic working characteristics of a hydraulic support.
The primary innovations in this paper are as follows: ( 1) The micro-dynamic model of a hydraulic support and the caving face roof and floor contact based on G-W contact theory is innovatively constructed, and an accurate contact stiffness measurement is obtained for the first time through theoretical deduction, which provides an accurate simulation parameter se ing method for simulation research.(2) A rigid-flexible-mechanical-hydraulic coupling dynamic model of a hydraulic support and the caving face roof and floor is established, and the accurate simulation of the interaction characteristics between a hydraulic support and the caving face is realized.
(3) A dynamic simulation system for the horizontal forward movement of the hydraulic support in industry is established for the first time, which reveals the dynamic working characteristics of the support moving from the perspective of macro-and microcharacteristics. (4) A comparative analysis of response characteristics of the support moving with or without pressure is carried out, which provides theoretical support for selecting support moving mode. (5)The process of lifting, moving, and lowering the hydraulic support is studied systematically, and its dynamic working characteristics are studied comprehensively.
The detailed work of this paper is as follows.In Section 2, the rigid-flexible coupling dynamic simulation model of a hydraulic support is established and the dynamic working characteristics of a hydraulic support lifting support are studied through this system.Section 3 studies the contact theory of a hydraulic support base and caving face floor and an equivalent stiffness theory of hydraulic cylinder based on G-W theory, and establishes a rigid-flexible-mechanical-hydraulic coupling dynamic simulation system of a hydraulic support and the caving face roof and floor, and studies the dynamic working characteristics of a hydraulic support moving.Section 4 studies the dynamic working characteristics of a hydraulic support with pressure, and compares the response characteristics of a hydraulic support with and without pressure.Section 5 gives some conclusions.
Establishment of Rigid-Flexible Coupling Dynamics Simulation Model for Hydraulic Support
Figure 1 displays the caving coal hydraulic support by the top beam, shield beam, tail beam, base, and other parts, for the purposes of modifying the hydraulic support components' response to be closer to the actual conditions, with the use of the Hypermesh pre-processing software package for hydraulic support flexibility.The material of support structure is defined as structural steel with density of 7860 kg/m 3 , Young's modulus of 2.1 × 10 11 Pa, and Poisson's ratio of 0.3, and the rigid node area set up at the hinge joint of the rotating pair is designed to enhance force transmission at the hinge point [36].To facilitate the lifting support motion, additional column drives have been installed on the front and rear columns.The simulation environment is as follows: ADAMS-2020 version is used for simulation, dynamic analysis type is default, dynamic integration solver is GSTIFF, dynamic integration format I3, dynamic allowable error is 0.001, kinematic allowable error is 0.0001, timestep is 0.001 s, optimization algorithm is MSCADS-MMFD, optimization tolerance is 0.001, optimization maximum iteration number is 100, optimization forward differentiation, Jacobi form integrator type is T:F:F:T: F:F:T:F, initial condition tolerance is 1 × 10 −10 , contact surface tolerance is 300, linear solver type is automatic, linear solver stability is 0.01.
Experiment of Hydraulic Support Lifting under Different Lifting Heights
To understand the influence of different lifting and lowering heights of support on its dynamic working characteristics, the front and rear columns are driven to descend by 30 mm, 45 mm, 60 mm, 75 mm, and 90 mm at a speed of 30 mm/s, and the support is lowered twice and lifted twice respectively.The hydraulic support starts at a height of 2500 mm, and the top outside of the top beam runs parallel to the bottom surface of the base.
Experiment of Hydraulic Support Lifting under Different Lifting Speeds
To investigate the impact of varying lifting and lowering speeds on the dynamic operational a ributes of the support, the front and rear columns were operated to lower the support twice and subsequently lift it twice.This was performed at lifting speeds of 10 mm per second, 20 mm per second, 30 mm per second, 40 mm per second, and 50 mm per second, with a consistent single movement magnitude of 60 mm.
Study on Microscopic Contact Theory between Hydraulic Support and Roof and Floor of Caving Face
In the context of the caving face, when the support comes into interaction with the caving face, their interaction is modeled as a rigid surface meeting a rough surface based on the G-W theoretical model.Each micro-convex body on the surface is considered independent, without influencing one another, and all are treated as spherical micro-convex bodies with identical curvature radius [37].The heights of the micro-convex bodies are distributed according to a Gaussian distribution.The contact model can be seen in Figure 2.Under the assumption that the distance between two planes is denoted as h, and neglecting the elastic interaction between the micro-convex bodies, if the height of a microconvex body is greater than h, it will be in contact with the rigid plane.The depth of indentation for a micro-convex body with height z can be calculated as d = z − h.Based on Hertzian contact theory, the contact behavior between these surfaces can be further analyzed: Contact area of individual micro-convex body: Force on individual micro-convex body: 3 Contact stiffness of individual micro-convex body: According to Her ian contact theory, the total number of micro-convex body in contact is where: N represents the total number of micro-convex bodies, is a density function of contact area of micro-convex body, n A is the nominal contact area, ( ) is the dissemination function of height z of convex body satisfying Gaussian distribution, E is the modulus of elasticity of rough surface.
From Equations ( 1)-( 4), by integrating over all micro-convex bodies, we obtain: Total contact area Total contact force Total contact stiffness
Construction of Rigid-Flexible-Mechanical-Hydraulic Coupling Simulation Model of Hydraulic Support and Floor of Caving Face
During the hydraulic support operation, the compression of the emulsion in the column cylinder leads to the manifestation of elastic properties as a result of applied force [38].To enhance the rigid-flexible coupling dynamics simulation model of a hydraulic support depicted in Figure 1, the column is substituted with a spring damping system.Table 1 illustrates the structural a ributes of the hydraulic cylinder and the physical properties of the emulsion.The total equivalent stiffness of the hydraulic cylinder can be obtained from Equation (8).Following this, the relationship between the equivalent stiffness of the hydraulic cylinder and the support height is established, completing the construction of the spring damping system.In Equation ( 8), the total equivalent stiffness of the hydraulic cylinder is the sum of the equivalent stiffness ka of the oil in the rodless cavity and the equivalent stiffness kb of the oil in the rod cavity, as shown below: The equation variables are defined as follows: ka-equivalent stiffness of oil in rodless chamber; kb-equivalent stiffness of oil in rod chamber; A1-cross-sectional area of rodless chamber piston in cylinder; A2-cross-sectional area of rod chamber in cylinder; L-effective stroke of hydraulic cylinder; L1-current fluid level in cylinder rodless chamber; xvibration displacement of system; V1-volume of oil in rodless chamber of hydraulic cylinder; V2-volume of fluid in cylinder rod chamber; Va-volume of oil in hydraulic line of rodless chamber in cylinder; Vb-the volume of fluid in a hydraulic line with a rod chamber in a hydraulic cylinder.
A cuboid entity is generated under the hydraulic support base as caving face floor, and the material is gangue, the density is 2800 kg/m 3 , the elastic modulus is 34 GPa, and the Poisson ratio is 0.3.Set the floor of caving face and hydraulic support base as collision contact, reasonably set the contact stiffness according to formula, complete the hydraulic support and caving face floor rigid-flexible-mechanical-hydraulic coupling simulation model construction [39,40], as shown in Figure 3, and realize the hydraulic support moving action by driving the pushing hydraulic cylinder.
Experiment of Hydraulic Support Moving under Different Moving Speeds
In order to study the dynamic working characteristics of a hydraulic support at different moving speeds, the hydraulic support was moved at speeds of 100 mm per second, 200 mm per second, 300 mm per second, 400 mm per second, and 500 mm per second, respectively, at the working height of 2500 mm.In order to make the hydraulic support move from stable state, the hydraulic support was set to begin moving by the second, and the moving distance was 600 mm.
Experiment of Hydraulic Support Moving under Different Moving Heights
In order to study the dynamic working characteristics of a hydraulic support at different moving speeds, under the working height of 100 mm/s, the support was moved at the support heights of 2200 mm, 2300 mm, 2400 mm, 2500 mm and 2600 mm respectively.The moving action was set begin moving by the second, and the moving distance was 600 mm.
Experiment of Hydraulic Support Moving under Different Floor Conditions
When the hydraulic support is moving, the hydraulic support base contacts and rubs against the caving face floor, and there may be some floating coal that has not been cleaned up on the caving face floor.In order to explore the effect of the caving face floor conditions on the support movement, the caving face floor is divided into three categories: ① If the floating coal on the floor is cleaned thoroughly, the support will make contact with the floor gangue directly.② If the floating coal on the floor is not cleaned thoroughly and some floating coal remains, the support will make contact with the coal gangue mixture, assuming that coal and gangue have the same influence on the contact conditions.③ If there is a lot of floating coal on the floor, the support base is in direct contact with the coal.The material characteristics of coal, gangue, and support structure are shown in the table.According to the material characteristics shown in Table 2, contact stiffness for three types of contact conditions was calculated by the contact theory formula, and friction force parameters were displayed in Table 3.The support moving height of 2500 mm and the support moving speed of 100 mm/s are adopted to carry out the support moving simulation experiment, and the equivalent contact stiffness and friction coefficient under three kinds of floor contact conditions are adopted respectively.
Dynamic Working Characteristics of Hydraulic Support Moving with Pressure
For unstable or easily broken roof, roof instability may occur during the short working cycle of support lowering, moving, and lifting.The dynamic characteristics of support under varying working conditions are studied to investigate the effects of pressure on its performance.
Construction of Rigid-Flexible-Mechanical-Hydraulic Coupling Model of Hydraulic Support and Caving Face Roof and Floor
To investigate the dynamic operational characteristics of a hydraulic support during the process of pressure-induced support movement, a simulation roof is constructed above the top beam using a rigid-flexible-mechanical-hydraulic coupled simulation model of hydraulic support and caving face floor, as depicted in Figure 4.The material being simulated is coal [41].A translational connection is established between the earth and the roof, with the translation track aligned with the force of gravity.
Experiment of Hydraulic Support Moving with Pressure under Different Heights
For the purpose of studying the influence of support moving height on support moving with pressure, support moving simulation experiments were carried out at support moving speed of 100 mm/s and residual working resistance of 500 kN at support moving heights of 2200 mm, 2300 mm, 2400 mm, 2500 mm and 2600 mm, respectively [42].
Experiment of Hydraulic Support Moving with Pressure under Different Speeds
For the purpose of studying the influence of support moving height on support moving with pressure, support moving simulation experiments were carried out at support moving height of 2500 mm and residual working resistance of 500 kN at support moving speeds of 100 mm per second, 200 mm per second, 300 mm per second, 400 mm per second, and 500 mm per second [43].
Experiment of Hydraulic Support Moving under Different Residual Working Resistance
In order to study and compare the influence of different load conditions on the working characteristics of a hydraulic support with pressure, under the conditions of support height of 2500 mm and speed of 100 mm/s, these experiments were carried out under different load conditions, with residual working resistances of 500 kN, 1000 kN, 1500 kN, 2000 kN, and 2500 kN, respectively.
Experiment of Hydraulic Support Moving with Pressure under Different Floor Conditions
With the aim of studying the effect of changed floor contact conditions on the work characteristics of a hydraulic support moving with pressure, simulation experiments were carried out with the equivalent contact stiffness and friction coefficient under three different floor contact conditions under the working conditions of residual working resistance of 500 kN at a moving height of 2500 mm and a moving speed of 100 mm/s.
Experiment of Hydraulic Support Moving with Pressure under Impact Load
In order to study that working characteristic of support under impact load, the support was driven to move with pressure under the working conditions of height 2500 mm, speed 100 mm/s, and working resistance 2500 kN, and the moving distance was 600 mm.Impact loads were applied 3 s after support transfer; impact loads were 250 kN, 500 kN, 750 kN, 1000 kN, 1250 kN, 1500 kN, 1750 kN, and 2000 kN, respectively; and impact time was 0.01 s.
Results of Hydraulic Support Lifting Experiments
The angular acceleration of the hinge point of the four-bar linkage obtained from the simulation experiment of the lifting support is shown in Figure 5, and the response force diagram of the front column and the rear column is shown in Figure 6.Meanwhile, in order to describe the difference in dynamic response stability more clearly, the standard deviation is used to describe the fluctuation degree of response.In order to make the description of results more intuitive, the fluctuation coefficient FC is introduced to represent the standard deviation.The standard deviation calculation formula is shown in Equation (9).
where n is the total number of all data in the sample and u is the average of all data in the sample.The larger the fluctuation coefficient λ, the greater the degree of fluctuation in the stable response, indicating poorer stability.Conversely, the smaller fluctuation coefficient λ on behalf of the smaller response fluctuation degree, the be er the stability.According to Figure 5, with the height of the lifting support decreasing, it is noticeable that the peak value of each hinge point angular acceleration of the four-bar linkage displays a clear increase, and the fluctuation frequency is higher.When the height of lifting decreases from 45 mm to 30 mm, the angular acceleration changes most obviously.
According to the alterations in peak response force and fluctuation coefficient of the front column in Figure 6a, by increasing the height of the lifting support, it is noticeable that the peak response force and fluctuation coefficient of the front column exhibit a nonmonotonic trend.Initially, they decrease, followed by an increase.When the lifting support height transitions from 30 mm to 60 mm, both the peak value of the front column response force and the fluctuation coefficient decrease, with the rate of decrease diminishing as the height increases.Subsequently, as the lifting support height changes from 60 mm to 90 mm, the peak value of the front column response force and the fluctuation coefficient start to rise, with the rate of increase intensifying as the height increases.According to the variation in peak response force and fluctuation coefficient of the rear column in Figure 6b, it can be found that with the increase in the height of the lifting support, the peak response force and fluctuation coefficient of the rear column present a monotonic decreasing trend, and the decreasing amplitude decreases with the increase in height.
Through the experiment of a hydraulic support lifting under different speeds, the hinge point angular acceleration of the four-bar linkage is depicted in Figure 7, while the response force of both the front and rear columns can be observed in Figure 8.According to Figure 7, with the speed of the lifting support increasing, each hinge point's angular acceleration peak of the four-bar linkage significantly increases, and the fluctuation frequency is higher.
According to Figure 8a,b, the front and rear columns response forces exhibit similar trends, with the rear column demonstrating significantly higher response forces compared to the front column.The peak response force and wave coefficient in the front column escalates with the lifting support speed, showing a linear increase in amplitude with the speed.
The analysis results indicate that an increase in lifting speed will notably affect the stability of the hydraulic support four-bar linkage, the load stability of the front and rear columns, and the peak response force.Moreover, this impact becomes more pronounced at higher speeds.
Results of Hydraulic Support Moving Experiments
Through simulation experiments of a hydraulic support moving under different speeds, the forces of each hinge point of the four-bar linkage and columns of a hydraulic support are obtained as shown in Figures 9 and 10 Figure 9 shows the force variation in each hinge point in four-bar linkage of the support with the change under moving speed.It is apparent from the curve in the figure that as the support moving speed increases, the peak force value at each hinge point shows a significant rise, along with a notable increase in the fluctuation range.At the beginning of simulation, the hinge force fluctuates obviously at every speed, and then stabilizes rapidly, which is caused by the first contact between the hydraulic support base and the caving face floor, which produces a large contact force.This process is the change stage from unbalanced state to stable state of the support.At the beginning and end of the moving support, the force at each hinge point increases obviously, and stabilizes after fluctuation.
The response force of Joint1 changes slightly at low speeds of 100 mm/s and 200 mm/s, and the response force decreases first and then increases during support moving.
In the process of the support moving, the response forces at Joint2 and Joint3 demonstrate a consistent decline trend, and unstable fluctuations occur in the joint force at the low speed of 100 mm/s and 200 mm/s.
It can be seen from Figure 10 that the peak response force and fluctuation range in the front and rear columns increase significantly when the moving speed increases.The response force in column fluctuates significantly at the beginning of simulation and at the beginning and end of the support moving.Unstable fluctuations of column response force occurs in the procedure of the support moving at low speed of 100 mm/s and 200 mm/s.In the procedure of the support moving, the response force in the front column increases while the force in the rear column decreases.
The acceleration curve of the top beam under different moving speeds is shown in Figure 11.When moving speed increases, the peak value and fluctuation range of top beam acceleration obviously increase.The acceleration of the top beam fluctuates obviously at the beginning of the simulation and at the beginning and end of the support movement.The response force in the column exhibits unstable fluctuations while transferring the support at both 100 mm/s and 200 mm/s low-speed conditions.During the support transfer beyond 100 mm/s, the acceleration of the top beam initially decreases and then increases.However, the change trend is not clearly evident when transferring the support at the low speed of 100 mm/s.
When the simulation of the support moving without pressure under each height and speed in sequence was completed, we obtained the reaction force at each pivot point, the reaction force at the column, and the acceleration of the top beam in the hydraulic support quadrilateral linkage.Because the change trend was not obvious enough from the time domain curve, in order to obtain the change characteristics accurately, we took the peak value and fluctuation coefficient of each group of data.The upper surface of the figure is the peak value.A scale is provided on the right side of the figure.The middle surface is the wave coefficient, using the Z axis scale.Since the middle surface is partially obscured by the upper surface, the color of the middle surface is mapped on the lower surface to assist in characterizing the wave coefficient.Figure 12 shows the characteristics of the hinge point response force, column response force, and top beam acceleration of the hydraulic support four-bar linkage under different support moving speeds and heights.Figure 12a-c show the force response characteristics of each hinge point of the hydraulic support four-bar linkage at different moving heights and speeds.From the diagram, it is evident that the stress variation pa erns of every hinge point in the four-bar linkage remain largely consistent.As the height of the moving support increase, the fluctuation coefficient of the hinge point force increases, while the peak value decreases.Similarly, as the speed of movement increases, both the peak response force and the fluctuation coefficient of each hinge point experience an increase.The height of the moving support significantly influences the peak response force of the hinge point when the support moves at a low speed, whereas its influence becomes marginal at high speeds.When the moving speed is 500 mm/s, the peak response force of Joint1 and Joint3 increases first and then decreases, and decreases first and then increases, with the increasing of moving height, but the change in amplitude is not large.When the height of the moving support is small, the speed of the moving support has a significant impact on the peak response force of the hinge point.However, when the height of the moving support is large, the peak response force of the hinge point becomes even more sensitive to changes in the speed of the moving support.On the contrary, the fluctuation coefficient of the hinge point is more sensitive to the height of the moving support when moving the support at high speed, and the fluctuation coefficient of the hinge point response force is more sensitive to the speed of the moving support when moving the support at a low position.
The column response force characteristics are shown in Figure 12d,e.The variation trend of the response force fluctuation coefficient of the front and rear columns is basically the same.With the increase in the support moving speed, the column response force fluctuation coefficient increases.The support moving height has a greater influence on the column response force peak value at low speed, while the influence is smaller at high speeds.The peak value of the column response force is more responsive to changes in the support movement height at low speeds.The fluctuation coefficient of the column force tends to increase with the support movement height, showing a greater sensitivity to height changes at high speeds and to speed changes at lower positions.With an increase in the support movement speed, the peak value of the front column response force rises, remaining relatively constant within the 100-300 mm/s range of the support movement heights.Within the 400-500 mm/s range, the movement speed initially decreases and then increases with the rising movement height.Conversely, the peak response force of the rear column decreases with the support movement height but increases with the movement speed.
The acceleration characteristics of the top beam during the support moving process are shown in Figure 12f.The peak acceleration and fluctuation coefficient of the top beam reach their maximum values at the height of 2600 mm and the moving speed of 500 mm/s.The peak value and fluctuation coefficient of the top beam acceleration increase with the speed of the moving support, and the peak value of the top beam acceleration increases with the height of the moving support, but the amplitude is less than that in line with the speed of the moving support.When the height of the moving support changes, the peak value and fluctuation coefficient of top beam acceleration are relatively stable, which proves that the acceleration response of the top beam is more sensitive to the speed of the moving support.
Through the simulation experiment of the hydraulic support moving under different floor conditions, the response parameters of the hydraulic support are obtained as shown in Figure 13.
According to the examination of the hydraulic support response results in Figure 13, it can be inferred that the peak value of the hydraulic support response has no obvious change under different floor contact conditions, while the response fluctuation coefficient is minimal under pure gangue floor contact conditions, and maximal under pure coal floor contact conditions, and the value under mixed coal gangue contact conditions is between the two.This indicates that with the increase in floating coal in floor, the response fluctuation in the hydraulic support moving is greater.
Results of Hydraulic Support Moving with Pressure Experiments
Through the simulation experiment of a hydraulic support moving with pressure under different height, various response parameters of the hydraulic support were obtained as shown in Figure 14.The response comparison between the support with pressure and without pressure under different moving speed is shown in Figure 14.The change trend is basically the same.The peak response force and fluctuation coefficient of the hydraulic support with pressure are greater than those of the hydraulic support without pressure.With the increase in moving height, the overall trend is increasing.The peak response force and the fluctuation coefficient change smoothly when the moving height increases from 2200 mm to 2400 mm, but increase significantly when the moving height increases from 2400 mm to 2600 mm.
Through the simulation experiment of the hydraulic support moving with pressure under different moving speeds, the response parameters of the hydraulic support were obtained as shown in Figure 15.The response comparison between the support with pressure and without pressure under different support moving speeds is shown in Figure 15, and its variation trend is basically the same.The peak response force and fluctuation coefficient when moving with pressure are greater than those when moving without pressure, and both show an increasing trend with the rise in moving speed.As the speed increases from 400 mm/s to 500 mm/s, the response force at Joint1 hinge point and the acceleration of the top beam show a notable increase.
Through the experiments of a hydraulic support moving with pressure under different residual working resistance, the response characteristics of the hydraulic support were obtained as shown in Figure 16.From Figure 16, it is evident that as the residual working resistance increases, the peak value and fluctuation coefficient of the response force at the hinge point of the fourbar linkage clearly increase.Furthermore, the peak value of the response force in the front and rear columns shows a significant increase, while its fluctuation coefficient exhibits no obvious change.The peak acceleration of top beam changes smoothly when the working resistance is 500-2000 kN, and increases greatly when the residual working resistance increases from 2000 kN to 2500 kN.The fluctuation coefficient of top beam acceleration diminishes first and then rises with the grow of working resistance, the minimum value appears at the working resistance of 1000 kN, and the maximum value appears at 2500 kN.The analysis results show that residual working resistance has a significant effect on the stability of the support moving with pressure, and proper residual working resistance should be carefully Through the simulation experiments of the hydraulic support moving with pressure under different contact conditions of floor, the response parameters of the support were obtained as shown in Figure 17.It can be seen from Figure 17 that the peak value of the hydraulic support response moving with pressure has no obvious change under different floor contact conditions, while the response fluctuation coefficient is the smallest under pure gangue floor contact conditions, and the largest under pure coal floor contact conditions, and the value under mixed coal gangue contact conditions is between the two.This indicates that the response fluctuation of the hydraulic support moving with pressure becomes larger with the increase in floating coal in floor.
Through the simulation experiment of the hydraulic support moving with pressure under the impact load, resulting in the impact response of the support depicted in Figure 18.In Figure 18, the peak response force and steady-state response force resulting from various impact loads during the movement of the hydraulic support with pressure are illustrated.The analysis indicates that both the peak response force and steady-state response force at the hinge point of the hydraulic support four-bar linkage increase linearly with the rise in the impact load.Similarly, the steady-state response force in the front and rear columns also exhibits a linear increase with the impact load.The peak response force demonstrates an increasing trend with noticeable fluctuations.Specifically, as the impact load increases from 1750 kN to 2000 kN, the peak response force experiences a substantial of 948.05 kN.These findings highlight the significant effect of the impact load on the stability of the support, with stability notably decreasing under higher impact loads.
Discussion
The analysis of the response of hydraulic support columns under an impact load in reference [14] is based on the ADAMS software package 2020, and the support components are treated with flexibility.Both this study and reference [14] used contact stiffness and penetration depth to calculate the contact force between the coal mining face and the support.Reference [14] does not explain how to calculate the contact stiffness.However, this study is based on the G-W contact theory and a micro dynamic theoretical model of the contact between the hydraulic support and the roof and floor of the caving face.For the first time, an accurate contact stiffness is obtained through theoretical derivation.Complex contact forces are generated by the contact between the hydraulic support and the roof and floor of the caving face instead of static loading to analyze the hydraulic support.Moreover, both this article and reference [14] treat the column as an equivalent spring damping system, but this article provides detailed calculations of the spring stiffness to further modify the simulation to be closer to actual working conditions.
Both this study and reference [14] studied the stability of hydraulic supports by analyzing the response force between the components and the hinge points of the hydraulic support.However, reference [14] only studied the hydraulic support under static load conditions, while this study considered the dynamic process of the hydraulic support and conducted in-depth research on various components with larger responses, obtaining comprehensive response parameters of the hydraulic support.
This study is based on the G-W contact theory and a micro dynamic theoretical model of the contact between the hydraulic support and the roof and floor of the caving face.For the first time, accurate surface contact stiffness is obtained through theoretical derivation.The complex contact force generated by the contact between the hydraulic support and the roof and floor of the caving face is used to analyze the hydraulic support instead of static loading.Reference [44] applied the Her theory to derive the contact force and contact stiffness of a rigid body in contact with a random elastic rough surface.The obtained expression is basically consistent with the results derived in this study.The direct derivation results in reference [44] are consistent with the three-dimensional simulation experimental results of rough surface contact in reference [45].
In summary, compared with reference [14], in a response analysis of a hydraulic support, this study establishes a micro-dynamic model based on G-W contact theory to accurately calculate the surface contact stiffness and calculate the equivalent spring stiffness of a hydraulic column in detail, so that the simulation is closer to the actual working conditions.Moreover, this study considers the dynamic processes and responses of multiple components more comprehensively than reference [14].In addition, the theoretical derivation results of this study are basically consistent with the Her theoretical derivation results in reference [44], and are consistent with the experimental verification in reference [45], further ensuring the reliability of the study.The above comparison indicates that the model construction method, research method, and simulation parameter se ing based on the theoretical research in this study have a scientific basis, and the results obtained are also reliable.
Conclusions
Based on the G-W contact theory, the paper innovatively puts forward a microcontact dynamic model of a hydraulic support and the caving face roof and floor, establishes a rigid-flexible-mechanical-hydraulic coupling dynamic simulation model of a hydraulic support and the caving face roof and floor, and obtains the following conclusions through a simulation analysis of lifting, lowering, and moving under various conditions: (1) With the increase in the height of the support lifting, the peak angular acceleration the hinge point of the four-bar linkage decreases, and the fluctuation frequency decreases; the response force of the front column decreases first and then increases, and the rear column decreases.The peak value and fluctuation of angular acceleration at the hinge point of the four-bar linkage increase with the increase in the speed of the lifting; the response force trend of the front and rear pillars is similar, but the rear pillar is obviously higher than the front pillar.In practical application, the relationship between the speed of the lifting and the stability of the system should be balanced, and the state of the rear column should be mainly detected.Hydraulic supports need to be designed with the height and speed of the lifting in mind to ensure that the support maintains stability and safety under various operating conditions. (2)The response of the hydraulic support is affected by different contact conditions of the floor, and the increase in floating coal on the floor will lead to a greater fluctuation in the response when moving the support.In actual fully mechanized coal mining, it is necessary to consider the influence of different floor conditions on the support stability, and it may be necessary to adjust the working parameters of the support to strengthen the adaptability to different floors.(3) The peak value and fluctuation of response stress are larger when the support is moving with pressure than when moving without pressure, and increase with the height and speed of the moving.This shows that the response of the structure will increase significantly due to the influence of an extra load when the hydraulic support is moving with pressure.When selecting the mode of the support moving with pressure, the working conditions should be carefully analyzed, and the speed and height should be controlled. (4)With the increase in the moving support height, the fluctuation of the hinge point force increases and the peak value decreases, and with the increase in the support moving speed, the peak value and fluctuation of the hinge point force increase.Under different height and speed conditions, the sensitivity of the response to the change in height or speed of the moving support is different.The height and speed of the moving support will affect the response of the hydraulic support structure obviously, so it is necessary to choose an appropriate height and speed of the support moving according to working conditions.(5) The increase in residual working resistance will lead to the increase in the response of the four-bar linkage and column, which will affect the stability of the support moving.The residual working resistance should be carefully selected. (6)When subjected to the impact load during the support moving, the response force of the four-bar linkage and column increases linearly and fluctuates obviously when the support is impacted.When designing hydraulic supports, it is necessary to consider the possible impact loads and take measures to enhance the impact resistance of hydraulic supports to ensure that they can maintain stability and safety when subjected to the impact load.
The research in this paper provides a theoretical model and accurate simulation parameter se ing method for the simulation research of surface contact between objects, provides an accurate rigid-flexible-mechanical-hydraulic coupling simulation model for the hydraulic support moving, provides a theoretical basis for the hydraulic support moving mode selection, and provides technical guidance for the hydraulic support design and optimization.
The main advantage of this research method lies in pu ing forward a micro-dynamic model of a hydraulic support and the caving face roof and floor based on G-W contact theory, establishing a rigid-flexible-mechanical-hydraulic coupling dynamic simulation model of a hydraulic support and the roof and floor of the caving face, and revealing the dynamic working characteristics of the support moving from the perspective of macroand micro-characteristics.This study systematically studied the lifting, moving, and lowering process of a hydraulic support, and comprehensively studied its dynamic working characteristics.The limitations of this research method lie using an equivalent spring damping system instead of a column, and using a driving kinematic pair instead of the simulation of a real hydraulic system, and when calculating the stiffness of the equivalent spring damping system, the influence of cylinder wall deformation and the entrapped bubbles in the hydraulic system is not considered, which leads to a certain gap between the simulation and the actual working conditions.
The future research directions are as follows: (1) further research on the control strategy and operation mode of a hydraulic support during lifting, lowering, and moving to improve the stability, safety, and efficiency of the mining face; (2) the combination of mechanical engineering, hydraulic pressure, control theory, and other disciplines to conduct comprehensive research and propose more comprehensive and systematic solutions to meet the requirements under different working conditions; (3) consideration of the real working conditions of a hydraulic system, including cylinder wall deformation, the entrapped bubbles, and other factors relating to hydraulic systems, thus improving the reliability of simulation.
Citation:
Xie, B.; Yang, Y. Study on Working Characteristics of 4-Column Hydraulic Support in Lifting-Lowering-Moving State Based on Microcontact Theory and Rigid-Flexible-Mechanical-Hydraulic Coupling Simulation
Figure 2 .
Figure 2. Contact model of rough surface.
Figure 3 .
Figure 3. Rigid-flexible-mechanical-hydraulic coupling simulation model of hydraulic support and caving face floor.
Figure 4 .
Figure 4. Rigid-flexible-mechanical-hydraulic coupling simulation model of hydraulic support and caving face.
Figure 11 .
Figure 11.Acceleration of top beam in simulation under different moving speeds.
Figure 12 .
Figure 12.Response of hydraulic support in simulation under different moving speeds and moving heights.(a) Response force of Joint1.(b) Response force of Joint2.(c) Response force of Joint3.(d) Response force of front column.(e) Response force of rear column.(f) Acceleration of top beam.
Figure 13 .
Figure 13.Response of hydraulic support in simulation under different floor contact conditions.(a) Peak.(b) Fluctuation coefficient.
Figure 14 .
Figure 14.Response of hydraulic support moving with and without pressure in simulation under different heights.(a) Response force of Joint1.(b) Response force of Joint2.(c) Response force of Joint3.(d) Response force of front column.(e) Response force of rear column.(f) Acceleration of top beam.
Figure 15 .
Figure 15.Response of hydraulic support moving with and without pressure in simulation under different speeds.(a) Response force of Joint1.(b) force of Joint2.(c) Response force of Joint3.(d) Response force of front column.(e) Response force of rear column.(f) Acceleration of top beam.
Figure 16 .
Figure 16.Response of hydraulic support in simulation under different residual working resistance.(a) Peak.(b) Fluctuation coefficient.
Figure 17 .
Figure 17.Response of hydraulic support moving with pressure in simulation under different floor conditions.(a) Peak.(b) Fluctuation coefficient.
Figure 18 .
Figure 18.Response of hydraulic support moving with pressure in simulation under different impact loads.(a) Peak.(b) Fluctuation coefficient. | 2024-05-22T15:11:38.871Z | 2024-05-20T00:00:00.000 | {
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30512687 | pes2o/s2orc | v3-fos-license | Physical determinants of asymmetric cell divisions in the early development of Caenorhabditis elegans
Asymmetric cell divisions are of fundamental importance for the development of multicellular organisms, e.g. for the generation of founder cells. Prime examples are asymmetric cell divisions in germline precursors during the early embryogenesis of the transparent roundworm Caenorhabditis elegans, one of the major developmental model organisms. However, due to a lack of quantitative data it has remained unclear how frequent unequal daughter cell sizes emerge in the worm’s early embryogenesis, and whether these originate from sterical or biochemical cues. Using quantitative light-sheet microscopy, we have found that about 40% of all cell divisions in C. elegans until gastrulation generate daughter cells with significantly different volumes. Removing the embryo’s rigid eggshell revealed asymmetric divisions in somatic cells to be primarily induced by steric effects. Division asymmetries in the germline remained unaltered and were correctly reproduced by a model based on a cell-size independent, eccentric displacement of the metaphase plate. Our data suggest that asymmetric cell divisions, imposed by physical determinants, are essential for establishing important cell-cell interactions that eventually fuel a successful embryogenesis.
cell volumes. As a consequence, extrapolated cell volumes are quite error-prone and may not report reliably on geometrical asymmetries in cell division events.
Despite these limitations, it is well established that at least cells of the future germline, the so-called P lineage (cf. the embryo's early lineage tree in Fig. 1A), undergo geometrically asymmetric divisions 2,12 . Yet, a thorough quantification of their (and other cells') asymmetries has, to the best of our knowledge, not been done. As a consequence, it is neither clear how many geometrically asymmetric cell divisions beyond the P lineage occur until gastrulation nor is it known what causes them. Indeed, one may even ask why C. elegans has geometrically asymmetric cell divisions at all since a biochemical asymmetry might have been sufficient to run the proper molecular-biological developmental program.
Here we have used selective plane illumination microscopy, SPIM, to address this topic (see, for example, refs 13-15 for introductory reviews on SPIM). Due to the gentle illumination via a light sheet, we were able to monitor the development of C. elegans embryos with and without an eggshell in three-dimensional detail up to gastrulation. A custom-made image segmentation approach enabled us to derive volumes and division asymmetries from these raw data. As a result, we observed that about 40% of all cell divisions before gastrulation are significantly asymmetric with many of these events being enhanced by sterical forces from the confining eggshell. For predominantly biochemically governed asymmetric cell divisions, i.e. for the P lineage, we were able to predict the degree of asymmetry via a simple model that relies on a cell-size independent, eccentric displacement of the mitotic spindle.
Results and Discussion
About 40% of all cell divisions until gastrulation are asymmetric. According to the literature, asymmetric cell divisions have been observed for cells of the P lineage (P 0 , …, P 3 ) and EMS 2,12,16 . These cell divisions coincide with the emergence of so-called founder cells (AB, MS, E, C, D, P 4 ) that establish new lineages (Fig. 1A). All other cell divisions until gastrulation are typically interpreted as being symmetric with respect to daughter cell sizes. In fact, data on volumetric asymmetries of daughter cells are mostly qualitative or extrapolated from two-dimensional imaging techniques rather than reporting faithful three-dimensional quantifications.
In order to obtain more quantitative insights into the amount and degree of volumetric asymmetries in cell divisions during early embryogenesis of C. elegans, we used a custom-made SPIM setup [17][18][19] and a custom-written segmentation approach (see Materials and Methods for details). The chosen worm strain (OD95) stably expressed fluorescent markers for histones (H2B::mCherry) and the plasma membrane (PH(PLC1δ1)::GFP), hence facilitating three-dimensional imaging and volume rendering during early embryogenesis. Representative examples of images and segmentation results are shown in Fig. 1B-D.
Using this approach we were able to identify all cell division events and to quantify all cell volumes until the onset of gastrulation via temporally resolved three-dimensional image stacks. Using these data as experimental input, we calculated for each mother cell the volume ratio of daughter cells, VR = V 1 /V 2 . For somatic cells we used the more posterior cell for V 2 , for germline cells always the (smaller) new germline cell volume was used for V 2 . To explore whether a division was significantly asymmetric, i.e. whether the value of VR deviated sufficiently from unity, we defined a level of uncertainty that arises solely from segmentation errors (see Materials and Methods for details). Roughly speaking, relative deviations of daughter cell volumes by 10% or more indicated a significant volumetric asymmetry in the respective cell division. The resulting data and their significance rating are shown in Fig. 1E.
As expected, all cells of the P lineage showed a significant division asymmetry, albeit with markedly different VR values (Fig. 1E). Also EMS was found to divide asymmetrically, although the asymmetry was less than for any cell of the P lineage. Surprisingly, also other cells at this early stage, namely MSa, MSp, Ca, and Cp, showed significant asymmetries with daughter cells differing by 30-60% in volume. In contrast, E, MS, and C divided almost perfectly symmetrical like most cells from the AB lineage. Yet, even some cells from the AB lineage showed a significant but borderline asymmetry, especially ABar.
Thus, our data indicate that altogether about 40% of all cell divisions until gastrulation in C. elegans embryos feature a significant volumetric asymmetry.
Geometrical constraints induce asymmetric divisions. Since, to our knowledge, a biochemical asymmetry in the cell division of MSa, MSp, Ca, and Cp has not been described, we hypothesized that at least some of the observed volumetric asymmetries might arise from geometric constraints during the respective cell divisions rather than being governed by biochemical cues. Indeed, geometrical constraints and physical forces have been seen to have a significant impact on the positioning of cells in the early embryo 17,18 but also in the context of tissue organization and wound healing 20,21 , making this hypothesis an attractive option.
In order to test our hypothesis, we sought to decrease possible mechanical constraints by removing the chitin-based eggshell, while leaving the inner, more flexible vitellin layer intact to maintain the embryo's integrity (see Materials and Methods). As expected, in the absence of the eggshell the intact vitellin layer lead to a rather compact arrangement of the blastomeres that was often, but not always, similar to wild-type embryos ( Fig. 2A). In fact, developmental phenotypes became significantly more variable: The stereotypical diamond-shape arrangement of cells in the four-cell state (Fig. 1B, second image) was seen only in about 50% of all embryos without an eggshell, whereas the remaining embryos displayed arrangements that resembled a (partially skewed) T-shape ( Fig. 2A, second image). These early deviations from the wild-type phenotype correlated with increasingly severe misarrangements in later stages, hence underlining the supportive role of the eggshell for a robust embryogenesis.
The volume ratios associated with eggshell-free embryos (Fig. 2B) and the direct comparison to untreated embryos ( Fig. 2C and Fig. S2) highlighted that the P lineage (P 0 to P 3 ) as well as Ca did not change markedly. This suggests that their volumetrically asymmetric divisions are a consequence of an internal biochemical asymmetry. Almost all other volume ratios showed a clear tendency to decrease, indicating that sterical forces induced by the confining eggshell cause at least partially these asymmetries. In particular, EMS and the MS lineage showed Representative maximum-intensity projections of image stacks taken on early C. elegans embryos (strain OD95) with the plasma membrane and chromatin stained in red and green, respectively. Scale bar: 10 μm. (C) Single two-dimensional slices taken from the image stacks shown in A. (D) The corresponding membrane segmentation shows how well details of the plasma membrane are identified. Please note: Color-coding of cell boundaries was chosen for best contrast and does not indicate correspondence to specific lineages. (E) Volumetric ratio, VR, of daughter cells emerging from the named mother cell (median of n = 10 embryos with error bars indicating the standard deviation). Color-coding of lineages like in (A). The volume-dependent level of uncertainty for each cell (grey) quantifies the apparent division asymmetry that is attributed solely to segmentation errors (see Materials and Methods for a detailed definition). As a result, cells of the P, MS, and C lineages, but also few cells of the AB lineage show significant division asymmetries that are well beyond the level of uncertainty. markedly reduced asymmetries, making them almost as borderline as the somatic outlier ABar. Cp showed a significantly reduced value of VR while still dividing in significantly asymmetric fashion, whereas Ca was almost unchanged. Thus, asymmetric divisions in the P and C lineages are well preserved even with softened geometric constraints while the asymmetry in EMS and in the MS lineage seem to rely predominantly on sterical forces imposed at least indirectly by the eggshell.
A cell-size independent displacement of the mitotic spindle quantitatively explains division asymmetries in the germline. Inspired by previous work 5, 6 on the first cell division in C. elegans embryos, in which a pronounced shift of the mitotic spindle apparatus along the AP-axis has been identified as major cause for an asymmetric cell division of P 0 , we hypothesized that also subsequent asymmetric cell divisions in the germline are driven by a displacement of the spindle's center of mass. In particular, we wondered to which extent a shift of the mitotic spindle could quantitatively explain the experimentally observed volumetric ratios VR of daughter cells in the post-zygote germline. For this analysis, we deliberately excluded P 0 , since several molecular players that influence an eccentric spindle displacement in P 0 from the anterior side 22,23 are segregated into the AB cell during the first cell division. Since gene expression is mostly shut down in the germline during early development 24 , these molecular players are therefore unlikely to play a major role in subsequent cell divisions in the germline, rendering the first division a special case (see also discussion below). Moreover, we have used data from embryos without eggshell for the subsequent analysis since these display asymmetric divisions without the influence of eggshell-induced cues; we did not observe significant differences when using data from untreated embryos.
For simplicity we assumed the mother cell to be spherical (radius R 0 ), and we asked how the volumetric ratio would be if the daughter cells emerged from a spindle that was displaced by an increment Δx away from the cell center (Fig. 3A, inset). Given that the position of the metaphase plate defines the plane of cytokinesis 25 , the spherical mother cell would be split approximately into two spherical caps with heights R 0 + Δx and R 0 − Δx, yielding volumes respectively. The mathematically simplest assumption for this division scheme would be a constant shift Δx that does not depend on the size of the mother cell.
To compare this naïve approach with our experimental data, we used the measured volume of P 1 from which we extracted the apparent cell radius via R 0 = (3 V/4π) 1/3 . Then, we iteratively predicted from this single experimental input the volumes V theo of all subsequent daughter cells along the lineage tree (P 2 , EMS, P 3 , C, P 4 and D): Volumes of EMS and P 2 , i.e. V 1 and V 2 , were derived via the spherical-cap scheme outlined above, using R 0 of P 1 as input. Assuming P 2 to be spherical again, we extracted its apparent radius from the predicted volume and repeated the division scheme until all volumes had been determined. The predictions we got from this procedure showed a remarkably good agreement with the experimentally observed volumes of daughter cells, V exp , when setting Δx ≈ 1.75μm (Fig. 3A). Using instead a shift Δx that depended on the mother cell size did not capture the experimental data.
Thus, a cell-size independent shift of the mitotic spindle by approximately 1.75 μm can quantitatively explain all experimentally observed volumetric division asymmetries in the post-zygote germline.
Next we sought to obtain experimental support for this simple approach and its prediction of a cell-independent displacement of the mitotic spindle by Δx ≈ 1.75μm during asymmetric division events in cells P 1 -P 3 . In contrast to the distinct first cell division, division axes of cells P 1 -P 3 do not necessarily lie in a single imaging plane, which virtually eliminates the possibility to determine the spindles' shift via very rapid two-dimensional imaging. We therefore utilized our three-dimensional image stacks, acquired with a moderate time resolution, as a proxy.
In particular, we exploited the last image stacks in which P 1 , P 2 , and P 3 showed an unambiguous metaphase pattern. Combining segmented image stacks and tracking data of chromatin, we determined position and orientation of the metaphase plate in the respective cell. As a reference point, we also calculated the cell center according to a mechanism proposed by Grill and Hyman (see Materials and Methods and Fig. 2c in ref. 26): Astral microtubules, docked to the cell cortex via special sites that are equi-distributed on the plasma membrane, exert pulling forces on the spindle, with the magnitude of force being independent of microtubule length. The equilibrium position determined by this mechanism is equal to the surface's center of mass for symmetrical cells but can deviate for unsymmetrical or non-convex cells. Assuming the plane of division to coincide with the metaphase plate, we determined the distance of the cell center from the metaphase plate along its surface normal as an estimate for the eccentric displacement of the spindle. Displacements were assigned a direction with negative/ positive signs indicating a shift towards the larger/smaller daughter cell.
The analysis outlined above was applied to all eggshell-free embryos (n = 11) from which median values and standard errors for each cell along the lineage tree were determined. As a result, we observed that displacements in somatic cells did not show a clear trend (Fig. 3B). In line with this, a Jarque-Bera test rated these data to be consistent with values drawn randomly from a normal distribution (mean: −0.1 µm; standard deviation: 0.56 µm; p = 0.5). Moreover, a very small correlation coefficient of 0.009 between these spindle displacements and the respective cell division asymmetries further supported the notion that somatic asymmetries are not a consequence of displaced mitotic spindles. This is in stark contrast to consistent spindle displacements in germline cells that were seen to cluster around a median value of 1.36 µm (Fig. 3B, red points). Moreover, a Kolmogorov-Smirnov test revealed a significance of 0.38% that displacement values of germline and somatic cells belong to the same distribution, i.e. it is extremely unlikely that the observed shifts in germline cells are only statistical fluctuations.
The observed shift in germline cells is clearly nonzero but somewhat lower than the predicted value, Δx ≈ 1.75μm. We attributed this difference to the fairly long lag time of ΔT = 30 s between successive image stacks, i.e. even after the last stack with a metaphase phenotype the spindle could still be on the move for up to 30 s. Based on data acquired for P 0 a peak velocity in the range of 40 nm/s can be expected for the spindle motion 6 . Assuming that all spindles in the post-zygote germline move with a somewhat lower, average velocity of 30 nm/s (see below for a justification) and estimating that the onset of cytokinesis happens on average ΔT/2 = 15 s after the image stack has been taken, an unmonitored distance of 15 s × 30 nm/s = 450 nm should be taken into account to extrapolate the typical shift of the spindle in cells P 1 -P 3 . The result, a shift by Δx≈1.36μm + 450 nm = 1.81μm, is in favorable agreement with our prediction derived via the division scenario into spherical caps. (dashed vertical line). A Kolmogorov-Smirnov test yielded a significance level of 0.38% that data from somatic and germline cells are from the same distribution, i.e. they can be regarded as different with a high significance. (C) Division asymmetries VR, predicted for P 1 , P 2 , and P 3 on the basis of the last image stack that shows an unambiguous metaphase (grey bars), follow the experimental results for the daughter cells This extrapolation for germline percursor cells is further corroborated by the volumetric asymmetries, VR, determined from the very same segmented image stacks: Relying again on the last image stacks in which P 1 , P 2 , and P 3 showed an unambiguous metaphase pattern, the metaphase plate was used to determine the future division plane. All voxels of cells P 1 -P 3 were sorted into putative daughter cells (see Materials and Methods), i.e. real cells were dissected through the metaphase plate into slightly deformed spherical caps. Values of VR determined via this procedure indeed followed the trend of the experimental data for fully developed daughter cells (Fig. 3C), yet consistently underestimated the asymmetry (consistent with the somewhat too low spindle displacement Δx≈1.36μm determined from these images). Shifting the putative division plane by 450 nm along its surface normal to account for the unmonitored spindle movement between successive image stacks, we obtained a favorable agreement between the estimated and experimentally determined asymmetries (Fig. 3C).
Thus, the experimentally determined displacement of the metaphase plate in cells P 1 -P 3 confirms the reasoning of a cell-size independent eccentric position of the spindle before asymmetric division events, whereas a significant shift of the metaphase plate in the ensemble of somatic cells was not observed.
A simple model explains the constant spindle displacement. The above results raise the question of how cells actually ensure a size-independent spindle displacement to an eccentric position before entering anaphase. To arrive at a meaningful model, we started from experimental observations in the zygote: In P 0 , the anterior spindle pole is tethered and remains in a fixed position until the spindle is fully assembled 27 . Upon release of the tethering, the metaphase plate starts to displace to the posterior with a constant velocity 27 . While the anterior spindle pole is displaced only slightly to the posterior 27 , the posterior spindle pole is displaced significantly stronger 5 . Hence, concomitant to its displacement the spindle is stretched along its migration path. Moreover, laser ablation experiments in P 0 have revealed that astral microtubules provide the pulling forces for the metaphase plate's displacement 5,6,8,27 . These pulling forces are generated at the cell cortex, at which astral microtubules are in contact with force generator complexes that involve Gα, GPR-1/2, LIN-5 and dynein (see ref. 2 for a recent comprehensive review). A net force ratio of ~1.5 towards the posterior is achieved in P 0 by locally increasing pulling forces on the posterior cortex 5, 6, 8 but also by decreasing forces on the anterior cortex 22,23 . Using RNA interference to switch both poles to the same force generator phenotype lead to a vanishing net force on the spindle 6 , and consequently these embryos lacked the native spindle displacement towards the posterior end. When both poles were forced to assume an 'anterior' force generator phenotype, mitosis even was stalled in late metaphase due to a too low absolute force that was insufficient to initiate a rupturing of the spindle.
Combining these observations with our results, we can formulate a simple one-dimensional model for achieving a cell-size independent displacement of the spindle that precedes an asymmetric division (Fig. 3D): Forces acting on the anterior and posterior spindle poles, F A and F P , do not depend on the distance to the cell cortex as microtubules only transmit pulling forces that are created at the cortex. Due to a low residence time of each microtubule at the cortex, about 1-2 s (ref. 28), microtubules also do not contribute a memory-driven restoring force. Therefore, F A and F P can be modeled as constant forces. The spindle does not provide active forces for its displacement but needs to oppose the net stress F p + F A applied via the spindle poles. For simplicity, we model it as a Hookean element with spring constant k and equilibrium length L 0 . Since initial spindle lengths at early metaphase are almost cell-size independent at these early stages of embryogenesis 29 , we can assume L 0 to be approximately the same for all P cells. Upon stretching this spring beyond a limit L 0 + s max , the spindle is assumed to rupture. This assumption is based on the observation that pulling on both spindle poles with only the anterior force magnitude is insufficient to go from metaphase to anaphase, whereas bidirectional pulling with the posterior force magnitude allows for spindle rupturing and (symmetric) cell division 6 . Due to the low Reynolds number in cell biology problems 30 , the motion of the spindle and/or its poles can be described in the overdamped limit, i.e. we can neglect all inertia terms.
For convenience, we express the equations of motion in terms of the distances x A and x P that the anterior and posterior spindle poles assume over time with respect to their initial position. Initially, the two poles are separated by the equilibrium length L 0 of the unstressed spindle, i.e. x A (t = 0) = x P (t = 0) = 0. Upon releasing the tethering at time t = 0, the forces F A < F P act on the spindle poles and the spindle is stretched by a distance s = x P −x A . Hence, the equations of motion read for t > 0: (black bars) but consistently underestimate the asymmetry. In fact, using these image stacks and dissecting segmented cells into two caps via a division plane through the metaphase plate is consistent with a median spindle shift of only 1.36 µm (see Fig. 3B and main text). Accounting for an additional, unmonitored spindle shift by approximately 450 nm during the lag period between consecutive image stacks (see main text) and repeating the dissection scheme the extrapolated asymmetries (red bars) show a favorable agreement with our experimental data. (D) Spindle displacement by a constant offset Δx can be rationalized by assuming constant forces F A < F P that pull the spindle towards the anterior and posterior end of the cell, respectively. As a result, the spindle is stretched and its center of mass moves into the posterior direction. Stress resistance of the spindle is modeled via a passive Hookean element (spring constant k, resting length L 0 ) until a maximum extension is reached and the spindle ruptures at the onset of anaphase. See main text for details.
with γ denoting the effective friction coefficient for the spindle poles. Solving these coupled differential equations, one obtains x MP (t) = [x A (t) + x P (t)]/2 = (F P − F A )·t/(2γ) for the position of the metaphase plate, and s(t) = (F P + F A )/(2k)·(1 − exp(−2kt/γ)) for the extension of the stressed spindle. Upon reaching a maximum extension s max , the spindle ruptures and cytokinesis is initiated. The associated instant of time, T, is determined via the equation s max = s(T) = (F P + F A )/(2k)·(1 − exp(−2kT/γ)) from which one can infer the maximum travel distance of the spindle, Δx = x MP (T) = (F P − F A )·T/(2γ). Since neither the forces F A and F P nor the spindle parameters dependent on cell size, this model predicts a constant displacement of the mitotic spindle.
It is worth noting that the spindle displacement explicitly depends on the ratio of anterior and posterior pulling forces, F A /F P . In fact, adapting the aforementioned approach of spherical caps to the ellipsoidal zygote, a displacement of ~3 µm was needed to explain the asymmetry ratio VR for P 0 , which is significantly larger than the value Δx≈1.75μm for P 1 -P 3 . This apparent discrepancy can be rationalized when taking into account that in P 0 several molecular agents downstream of the PAR machinery are localized in the anterior domain of the cortex where they contribute to a lowering of pulling forces towards the anterior 22,23 . Assuming that the cytoplasmic pool of these proteins is much smaller than the pool localized on the anterior cortex 23 , the majority of these very proteins would be segregated into the somatic cell AB and hence was lost for the future germline. Since gene expression is mostly shut down in the germline at these early stages of development, it is unlikely that comparable levels of these proteins could be restored soon after the first cell division. As a consequence, these players would not be available to reduce F A in cells P 1 -P 3 as much as in P 0 , hence increasing the ratio F A /F P in these cells. This leads to a slower spindle displacement in P 1 -P 3 in comparison to P 0 (as assumed when estimating the unmonitored spindle shift by 450 nm), whereas the spindle-internal stress builds up more rapidly. Therefore, upon reaching its maximum extension s max , the spindle has travelled a smaller distance. Following this reasoning, cells P 1 -P 3 are predicted to display a smaller displacement Δx than P 0 , in favorable agreement with experimental observations. Finally, one may wonder why nature has chosen to equip C. elegans with volumetrically asymmetric cell divisions during early embryogenesis, as biochemical asymmetries could have been fully sufficient. While cell sizes seem to have little influence on the positioning of cells until gastrulation 17,18 , the number of cell-cell contact areas certainly depends quite strongly on the surface area of cells. We therefore speculate that distinct division asymmetries cause, or at least support, the formation and/or prevention of cell contact areas to achieve a wiring diagram of cells that can fuel a successful embryogenesis.
Materials and Methods
Sample preparation and imaging. For imaging we used C. elegans strain OD95 in which the plasma membrane and histones are fluorescently labeled (PH(PLC1δ1)::GFP, H2B::mCherry). Worm culture and preparation of untreated embryos for SPIM imaging was done as described before [17][18][19] . Removal of the eggshell was done similar to previous approaches 31 : Zygotes with visible pronuclei were chosen from dissected gravid worms. These eggs were placed on a coverslip and immersed in approximately 30 μl of NaOCL solution (3% Na) for two to three minutes. Subsequently they were washed three times with M9 buffer 32 to remove all of the NaOCL solution before pipetting 25 μl Chitinase solution onto them. The solution was prepared by dissolving 5 units of Chitinase from Streptomyces (Sigma) in 2 ml of sterile egg buffer 32 .
Removal of the eggshell took roughly 10 to 15 minutes. The remaining vitellin layer was left intact. When the eggshell was not visible any more, embryos were transferred rapidly to the SPIM setup for immediate imaging (starting in most cases during mitosis of the zygote). Embryos adhered to the plain, untreated glass surface without the need for Poly-L-lysine or other mounting agents. During imaging, unperturbed embryos were immersed in water, embryos without eggshell in M9 buffer. Imaging was performed with a custom-made dual color SPIM setup as described before [17][18][19] . For long-term imaging of wildtype and eggshell-free embryos, full dual-color stacks, consisting of 50 individual layers with a spacing of 2 μm, were taken every 30 s for a total time of three hours (i.e. 360 stacks).
Segmentation, image analysis, and evaluation. Tracking of nuclei via H2B::mCherry was done as described before 17 . We generally tracked at least until the embryo consisted of 44 cells and included a manual correction step to account for potential errors.
Three-dimensional segmentation of cell membranes from PH(PLC1δ1)::GFP images required a refined approach to account for SPIM-inherent shadowing effects. These arise from absorption and scattering events at bright structures when being illuminated by the light sheet, i.e. some shadowing is observed behind such structures. As a result, image segmentation of membrane-labeled embryos via global filtering and thresholding operators was not reliable. We have therefore developed a novel segmentation algorithm that is based on growing a seed region in each cell: The goal of the segmentation is a division of a three-dimensional image of an embryo with n cells into n + 1 regions, with each voxel of the image stack being uniquely assigned to one of the cells or to none ( = outside of the embryo). After an initial box-filtering (kernel size 5 × 5 × 1 voxels) the background intensity of the image, i.e. anything outside of the embryo, is set to zero via global thresholding. Then, a seed is placed inside each cell, either manually or by using voxels that have been identified during the tracking of nuclei. During the segmentation process, these n seeds are grown simultaneously and iteratively. Boundaries of each seed are computed by eroding the region of voxels belonging to the seed with the smallest possible kernel (3 × 3 × 3 voxels) and subtracting this result from the original region. Boundary voxels therefore share one face, edge, or corner with voxels outside of this seed's region. Next, for each of these boundary voxels one neighbor outside the region is chosen randomly and both voxel intensities are compared. If the outside voxel's intensity is larger than the boundary voxel's intensity (F out ≥ qF boundary ), the outside voxel is added to the region unless it belongs already to another seed's region. The multiplier q ≈ 0.97…0.99 is introduced to compensate for noise, and it needs to be chosen carefully for each image or image series. This procedure is carried out for all cells/seed regions prior to the next iteration.
Aiming at short processing durations, a total of N = 300/log(n + 1) iterations were performed initially on a downscaled version of the image stack. After upscaling to the original size, 40 additional iterations were performed.
This scheme leads to a local expansion of each seed until it collides with another region or when its boundary arrives at a significant drop in voxel intensity. The latter typically occurs at the cytoplasm-membrane interface, i.e. the boundary of each region becomes a faithful representation of the plasma membrane. Minor artifacts (stray pixels etc.) are removed after the iteration process by opening and closing operations with a kernel of 15 × 15 × 3 voxels. Results were finally controlled manually stack by stack to ensure a high segmentation quality. Results obtained with this algorithm provided us with data of cellular volumes (and shapes) of unprecedented precision.
From the segmentation process, the number of cells, n, and, the number of voxels of each cell, m i (i = 1, …, n), is known for each embryo at each instant of time. From this, each cell's volume was determined as the product V i = L x L y L z m i with L x = L y = 0.16μm being determined via the objective and the camera sensor, and L z = 2 μm the spacing between two consecutive optical sections within an image stack. Volumes do not show any significant changes during the cell cycle, i.e. a cell's volume can be assumed constant (data not shown). For our analysis, we used the median value of the obtained time series of cell volumes to suppress few possible outliers in the scheme. As a result, the sum of volumes of the daughter cells generally deviated from the mother cell's volume by less than 3%, indicating reliable segmentation results throughout the whole image series.
Volume ratios of somatic daughter cells were defined as the volume of the more anterior cell divided by the volume of the more posterior cell, e.g. VR AB = V ABa /V ABp . For germline cells always the smaller volume of the new germline cell was used in the denominator, e.g. VR P3 = V D /V P4 . The level of uncertainty due to segmentation errors, i.e. the minimal ratio VR that reports a significant division asymmetry, was determined as follows: The main source of error during volume determination originates from the decision whether or not an additional layer of voxels around the already detected volume is considered while segmenting a cell, i.e. if the region is expanded even further or not. Assuming all cells to be spherical with radii being determined by the cell volume, π = R V (3 /4 ) 1/3 , we can express this additional volume ΔV in analytical terms. We first note that due to L z ≫ L x we have to separately consider the bottom and top slice in the (sub)stack containing the respective cell, i.e. we have two contributions, ΔV 1 and ΔV 2 , from the inner layers and the two spherical caps in the top and bottom layer, respectively. We reasoned that the true cell boundary will, on average, bisect the thickness of the top and bottom layers (cf. sketch in Fig. S1), i.e. top and bottom caps have a height h = L z /2 and a squared in-plane radius . The contribution from the inner layers, where additional voxels have an average width L x , is simply the volume of a spherical shell with thickness L x , reduced by the volume of the two caps that are situated in the top and bottom layer, . Since any of these additional volumes will contribute only by chance, adding or not adding these voxels has equal probability 50%, the average volume that is added or not considered amounts to ΔV=(ΔV 1 + ΔV 2 )/2. Hence, even a symmetric division into daughter cells with volume V sym can lead to an apparent asymmetry VR max = (V sym + ΔV)/(V sym − ΔV) > 1 or VR min = (V sym − ΔV)/(V sym + ΔV) < 1, depending on which daughter cell determines the ratio's (de)nominator. Based on this reasoning, we only deemed measured asymmetries as significant when they exceeded this mother-cell volume-dependent uncertainty range.
To estimate the spindle displacement in late metaphase and to extrapolate the future division plane, we used three consecutive stacks (named S 1 , …, S 3 ) after the last image stack that showed an unambiguous metaphase phenotype (named S 0 ). From stack S 0 , the center-of-mass position of the metaphase plate, r 0 , is known from tracking the histone stain (H2B::mCherry). The cell center, c, associated with a symmetric spindle position was determined from the plasma membrane segmentation according to an iterative mechanism proposed by Grill and Hyman 26 : First, we selected an evenly distributed subset of ~3000 voxels on the cell surface and used their coordinates p i as origins of forces. As a starting point for the iteration we used the center of mass of these points, c(0). This position was iterated by calculating the direction of the effective net force, f = Σ(c(j) − p i ) / |c(j) − p i |, with the sum running over all voxel indices. Then, c(j + 1) = c(j) + εf with a small, empirical coefficient ε. This step was repeated until the sequence of c(j) converged to a position c that did not change significantly any more. For symmetrical objects, c will be identical to the center of mass of the object's boundary, while for unsymmetric or non-convex objects like real cells a small but significant deviation remains.
Positions r 1 and r 2 of daughter-cell chromatin assemblies are known for S 1 , …, S 3 from tracking the histone stain (H2B::mCherry). Normalizing and averaging the vector r 2 − r 1 over stacks S 1 , …, S 3 yielded a robust estimate for the surface normal of the future division plane, d. The distance by which the metaphase plate had been shifted to an eccentric position in stack S 0 was then determined as the scalar product Δx = d·(c − r 0 ). Then, the position of the metaphase plate was split into two artificial points q 1 and q 2 along the direction of d with a small separation of 10 nm («L x ), i.e. q = r 0 ± d·5 nm. Based on the shortest distance to these points, all voxels of the cell were then classified to belong to q 1 or q 2 , and the resulting volumes V 1 and V 2 were used for Fig. 3A. Indeed, this scheme splits the volume of the mother cell into two parts along a plane (perpendicular to d) that runs through the last metaphase plate position, r 0 . When extrapolating the unmonitored spindle movement between stacks S 0 and S 1 , the same approach was used with points q 1 and q 2 being shifted by d·450 nm. | 2018-04-03T01:42:15.545Z | 2017-08-24T00:00:00.000 | {
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233604022 | pes2o/s2orc | v3-fos-license | Effect of Short-Term Exposure to Fine Particulate Matter and Temperature on Acute Myocardial Infarction in Korea
Background/Aim: Previous studies have suggested that the short-term ambient air pollution and temperature are associated with myocardial infarction. In this study, we aimed to conduct a time-series analysis to assess the impact of fine particulate matter (PM2.5) and temperature on acute myocardial infarction (AMI) among adults over 20 years of age in Korea by using the data from the Korean National Health Information Database (KNHID). Methods: The daily data of 192,567 AMI cases in Seoul were collected from the nationwide, population-based KNHID from 2005 to 2014. The monitoring data of ambient PM2.5 from the Seoul Research Institute of Public Health and Environment were also collected. A generalized additive model (GAM) that allowed for a quasi-Poisson distribution was used to analyze the effects of PM2.5 and temperature on the incidence of AMI. Results: The models with PM2.5 lag structures of lag 0 and 2-day averages of lag 0 and 1 (lag 01) showed significant associations with AMI (Relative risk [RR]: 1.011, CI: 1.003–1.020 for lag 0, RR: 1.010, CI: 1.000–1.020 for lag 01) after adjusting the covariates. Stratification analysis conducted in the cold season (October–April) and the warm season (May–September) showed a significant lag 0 effect for AMI cases in the cold season only. Conclusions: In conclusion, acute exposure to PM2.5 was significantly associated with AMI morbidity at lag 0 in Seoul, Korea. This increased risk was also observed at low temperatures.
Introduction
Acute myocardial infarction (AMI) is a leading cause of death worldwide [1,2]. Similar to other countries, the incidence of AMI in Korea has increased over the last few decades [3,4]. Therefore, it is important to prevent the occurrence of AMI and identify the risk factors of AMI.
Many environmental factors have been suggested as risk factors for AMI. Air pollution has been repeatedly associated with increased risks of hospital admissions and deaths due to cardiovascular disease. Specifically, particulate matter (PM) has been identified as a risk factor for cardiovascular disease in studies performed throughout the industrialized world [5][6][7]. Air pollution is increasing as urbanization and industrialization processes expand worldwide.
Recently, growing evidence has shown that fine particulate matter (PM2.5), which is ≤2.5 µm in aerodynamic diameter, may play a role in the development of cardiovascular diseases and that different sizes of particulate matter affect cardiovascular health differently [8][9][10]. Previous research suggested that although smoking is a more important risk factor for cardiovascular mortality, exposure to PM2.5 is also a risk factor for the disease via mechanisms including oxidative stress and systemic inflammation [5,11]. Some studies have also shown that both short-and long-term particulate matter exposure are specific triggers of myocardial infarction (MI) [5,6]. A multicity study in China examined the short-term effects of air pollution on AMI morbidity and found that air pollutants, including PM10, were positively associated with the daily AMI admissions at lag 2 and lag 3 days [12].
In addition to the PM2.5, the temperature can also be a risk factor for adverse cardiovascular outcomes. Previous studies suggested that high or low temperatures are associated with the AMI mortality and morbidity. For instance, studies conducted in Cuba, Sweden, Massachusetts and Denmark reported an increased AMI risk at low temperatures [13][14][15][16]). Other studies conducted in South Korea and England identified that both low and high temperatures are associated with an increased AMI risk [17,18].
South Korea has serious air pollution concerns due to the frequent haze events and PM2.5 concentrations in recent years [19,20]. Seoul is the largest city in South Korea and it is a highly urbanized area with many emission sources, such as automobile exhaust, industrial factories, and power-generating facilities [21]. Furthermore, because of local and regional emission, and meteorological and chemical interactions, there is a high concentration of PM2.5 in Seoul.
The temperature of South Korea is also unique when it is compared to the other countries. This is because South Korea has four distinct seasons and a relative wide temporal variation in climate. The weather of South Korea is different between the central and southern parts of the Korean peninsula, and the central part, including Seoul, is colder than the southern parts [17].
Therefore, it is very important to identify the effect of PM2.5 and temperature on acute myocardial infarction (AMI) events in Seoul, Korea, which is highly polluted by PM2.5. We conducted a population-based study designed to investigate the impact of PM2.5 and temperature on acute myocardial infarction (AMI) among adults over 20 years of age in Seoul, Korea, by using data from the Korean National Health Information Database (KNHID).
Data Source
This study used data from the Korean National Health Information Database (KN-HID) collected between 1 January 2005 and 31 December 2014. The KNHID contains information about participants who visited hospitals under the Korean National Health Insurance Service (NHIS) program [22]. Since this National Health Insurance Service in Korea is a single-payer program and is mandatory for all residents, the KNHIS represents the entire Korean population and can be utilized as a population-based database [23]. The KNHID includes five databases (an eligibility database, a national health screening database, a healthcare utilization database, a long-term care insurance database, and a medical institution database) and contains data on the diagnosis and status of outpatients and inpatients [23].
The monitoring data of ambient PM2.5 from the Seoul Research Institute of Public Health and Environment were also collected. In Korea, the city of Seoul has recognized the importance of exposure to PM2.5 and began measuring PM2.5 in early 2000. Daily PM2.5 values during the study period between 1 January 2005 and 31 December 2014, were taken from 25 monitoring sites installed in the administrative districts of Seoul using the SPM-613D beta gauge method [24]. The daily PM2.5 values in Seoul were computed by averaging the daily mean concentration of PM2.5 at all monitoring stations.
To estimate other co-pollutants, including CO, SO 2 , and NO 2 , we obtained complete air pollution data from local district air quality fixed-site monitoring stations in Korea managed by the National Ambient Air Monitoring System [25]. Because the data on air pollution were not available at all administrative sites in Seoul, we applied kriging models to derive exposure assessments using geographic information systems (GIS) tools (ArcGIS Version 9.3, ESRI, Redlands, CA, USA) to estimate ambient air pollution levels in unmonitored districts. In addition, kriging interpolation considered a semivariogram model, and to evaluate the performance of the kriging model, the root mean square standardized error was referred to. Daily values were computed by averaging the daily mean concentration of air pollutants at all administrative districts in Seoul.
Weather condition data, including daily average temperature, daily maximum temperature, daily minimum temperature, daily mean relative humidity, and dew-point temperature were acquired from the database of the Korea Meteorological Administration (KMA), which runs 76 automated weather stations in the Seoul metropolitan area.
Ethic Approval
This study was approved by the Institutional Review Board of Ewha Womans University Hospital, Seoul, Republic of Korea (IRB number: EUMC 2019-12-009).
AMI Events and Exposure Definition
We obtained the study population data from the KNHID between 1 January 2005 to 31 December 2014. The AMI patients were defined as persons who were newly diagnosed under a diagnostic code for AMI. Only the day of the first diagnosis of AMI was designated as a day with an AMI event. We identified the AMI diagnosis from the patient's medical treatment based on the Korean Classification of Diseases, 6th revision (KCD-6), which is a modified version of the International Classification of Disease, 10th revision (ICD-10). We set the criteria of AMI patients based on the KCD-6 code for acute myocardial infarction (codes I21). Data regarding 192,567 AMI events in Seoul were collected during the study period. For the temperature exposure, we used three daily temperatures including daily mean temperature, daily maximum temperature, and daily minimum temperature.
Statistical Analysis
A time-series design was used to analyze the daily data of AMI cases, PM2.5 concentration and weather variables that were linked by date. Since the variance of daily AMI cases is greater than the mean, the daily AMI cases assumed a quasi-Poisson distribution (Supplementary Table S1). We fit a generalized additive model (GAM) to identify the association between PM2.5 and AMI cases: where E(Y t ) represents the number of AMI events at day t; s shows smoothing spline and β represents the log-relative risk of AMI morbidity associated with a unit increase of PM2.5. Relative risks (RR) of AMI morbidity with a 10 µg/m 3 increase in PM2.5 concentration was calculated. To control for the seasonal patterns and long-term trends, we considered smoothing spline for calendar time with 4 degrees of freedom per year to control for seasonal trends, temperature with 6 degrees of freedom, dew point temperature with 3 degrees of freedom and relative humidity with 5 degrees of freedom [26]. The day of the week was controlled as a categorical variable. We used the value of temperature, dew point temperature, and relative humidity at lag 0 as a covariate. Degrees of freedom (df ) were further tested by the sensitivity analyses.
To figure out the relationship between ambient PM2.5 exposures and AMI morbidity, we fit the models with different lag structures from lag 0 days (at the day of the AMI diagnosis) to lag 3 days. To consider the exposure effects of the average over the same and previous days, we used the 2-day to 4-day moving averages of PM2.5 to estimate the association between PM2.5 exposure and AMI morbidity [27]. We also explored the effect of three daily temperatures on AMI morbidity in adults.
In the main model, we assumed that the relationship between exposure variables (i.e., PM 2.5 ) and AMI events was linear. However, the exposure-response relationship may also indicate non-linearity. Therefore, we visualized the exposure-response relationship between the exposure variables (PM 2.5 and temperature) and the AMI events.
The stratified analysis of PM 2.5 concentration and daily AMI events was further carried out in the cold months (October-April) and warm months (May-September) separately [28,29]. We also defined the daily mean temperature strata in four levels (<3.70 • C, 3.70-14.30 • C, 14.30-22.40 • C, and ≥22.40 • C) by using quartiles and examined the relationship within each group.
All p values were 2-sided, and those less than 0.05 were considered to be significant. The statistical analyses were performed using the R (version 3.5.2) 'mgcv' package (R development Core Team, Vienna, Austria). Table 1 shows the summary statistics of air-pollutant concentrations and meteorological indicators including daily temperature, relative humidity and dew point temperature. During the 10 years under study, the mean daily PM2.5 concentration was 25.7 µg/m 3 . The mean daily average temperature was 12.7 • C and the average daily maximum temperature was 17.1 • C. The average relative humidity was 60.6% during the study period. The concentrations of PM2.5 were higher in the cold season. The daily average temperature was 22.8 • C in the warm season and 5.39 • C in the cold season (Supplementary Table S2).
AMI Incidence
Supplementary Table S3 represents the newly diagnosed AMI patients enrolled in the NHID in the overall cities and Seoul in Korea from 2005 to 2014. Of the 192,567 AMI cases that occurred during the study period, 17.27% of the data were cases that occurred in Seoul.
The crude incidence in Seoul was lower than the overall crude incidence. In 2005, the incidence of AMI was the highest in Seoul. In 2005, the crude incidence per 100,000 was 46.4. In 2011, the crude incidence per 100,000 in Seoul was 39.3, which was the lowest incidence during the study period.
Relative Risk Estimates for Cases of AMI Events
RR and 95% CI regarding the relationship between 10 µg/m 3 , the increase in PM2.5 at different exposure days and daily cases of AMI are summarized in Table 2. In the single pollutant models, the models with lag structures of lag 0 and lag 01 showed significant associations with AMI (RR: 1.011, CI: 1.003-1.020 for lag 0, RR: 1.010, CI: 1.000-1.020 for lag 01). In the co-pollutant model which simultaneously included two pollutants (PM2.5 and alternatively NO2, SO2 or CO), we could observe significant associations between PM2.5 and AMI events in the lag 0 model only. Figure 1 presents the average exposure-response curve between PM2.5 concentrations and the risk of daily AMI events. There were exposure-response relationships of the PM2.5 concentration and AMI at lag 0, lag 1, and the 2-day averages of lags 0 and 1 (lag 01). We also noted a broadly linear association for the 3-day average of lags 0, 1 and 2 (lag 02). A longer lag association show relatively flat or negative curves at lag3 and lag 03. Table 2. Adjusted relative risk estimates for daily cases of AMI event (95% CI) per 10 µg/m 3 increase in PM2.5 at single-lag (lag 0, lag 1, lag 2 and lag 3) and cumulative-lag (lag 01, lag 02 and lag 03) models a . The exposure-response curve for the association between concentration of PM2.5 and AMI over lag days. The black line is the log relative risk, and the gray area is the 95% confidence intervals of the risk estimates. Figure 2 shows the nonlinear curve for the association between three different temperatures (mean temperature, minimum temperature, and maximum temperature) and AMI. The results show a non-linear positive relationship between mean temperature and AMI. Furthermore, if the maximum temperature level increased, the log relative risk of AMI also increased nonlinearly. However, when we assumed the linear effect of the temperatures and identified the relationship between the three daily temperatures and daily cases of AMI events, the results showed no significant relationships between temperatures and AMI (Supplementary Table S4). Table 3 shows the adjusted RR for daily cases of AMI events per 10 µg/m 3 increase in PM2.5 at different lag days, which is stratified by the quartile level of daily mean temperature at lag 0. The results showed when the daily mean temperature level at lag 0 was between 3.70 and 14.30 • C, a 10 µg/m 3 increase in PM2.5 at lag 0, lag 01 and lag 02 were significantly associated with increased daily cases of AMI events. In the co-pollutant models at Supplementary Table S6, PM2.5 at lag 0 were significantly associated with AMI events, and Lag 01 was only associated with AMI in SO2 adjusted model. In different temperature groups such as <3.70 • C, 14.30-22.40 • C, and ≥22.40, the results did not show any significant associations. Figure 3 shows the results of stratified analyses by season. The associations between PM2.5 concentration and the cases of AMI events varied by season. The strati-fied analysis carried out in the cold season (October-April) and the warm season (May-September) showed a significant lag0 effect for AMI cases only in the cold season (RR: 1.012, CI: 1.002-1.023) after the adjustment for the covariates. Except for the lag0 effect, the other lag effects were not significant. Figure 2. The nonlinear curve for the association between different temperatures and AMI. The black line is the log relative risk, and the gray area is the 95% confidence intervals of the risk estimates. * p < 0.05. a Results adjusted for calendar time, daily mean temperature, dew-point temperature, relative humidity and day of week. b Using the quartile of temperature as the cut-off value. Figure 3. Adjusted relative risk for daily cases of AMI events (95% CI) per 10 µg/m 3 increase in PM2.5 t single-lag (lag 0, lag 1, lag2 and lag3) and cumulative-lag (lag 01, lag 02 and lag 03) models according to the season a . a Results adjusted for calendar time, daily mean temperature, dew-point temperature, relative humidity and day of the week.
Discussion
This study suggests there is evidence of an association between PM2.5 concentration and AMI morbidity in adults at lag 0 and lag 01 in Seoul, Korea. However, the adverse effect of PM2.5 at lag01 became insignificant after adjustment for other co-pollutants. Our results are mostly consistent with previous studies that reported detrimental heart effects from short-term exposure to PM2.5. A cohort study performed in Beijing showed a significant association between PM2.5 concentration and ischemic heart disease (IHD) morbidity in lag 0 and lag 0-2 days [30], which is consistent with our study. Furthermore, a study conducted in Belgium, which has a lower level of particulate matter than Seoul, demonstrated a significant effect of PM10 on AMI events at lag day3. However, the unconstrained distributed lag model did not show a significant relationship [31].
In the stratified analyses by the season and daily mean temperature, the relationship was significant only in the cold season. Furthermore, when the daily mean temperature at lag 0 was between 3.70 and 14.30 • C, the increase of PM2.5 at lag 0, lag 01 showed significant positive associations with the daily cases of AMI events. In March and April, the mean daily temperature is 5.47 and 12.0 • C, and Asian dust from dust storms is common in Korea. Therefore, this seasonal phenomenon can be another factor contributing to cardiovascular events [32]. In the warm season, the PM2.5 concentration was not associated with the AMI morbidity. One possible reason for the significant association in the cold season is connected to a low temperature and increased blood pressure and viscosity in the cold season, which might be important causal factors in increasing winter morbidity due to heart attacks and strokes [33,34].
Our results indicate the effect estimates were higher in the cold season, but they did not show that if the temperature drops the risk increases since the daily mean temperature group <3.70 • C showed a negative relationship between PM2.5 increase and the risk of AMI. In very cold weather, people stay home or inside. This activity pattern might attenuate the effect of the lowest temperature group on AMI, and thus only the medium temperature group shows a significant positive association between PM2.5 and AMI events. A study of seasonal variations in hospital admissions with AMI in Korea also found that AMI events increased during October to December (daily mean temperature in October to December: −1.00 to 15.5 • C) and then reduced in January to February (daily mean temperature of January and February: −2.46 and 0.50 • C) [35].
Analyses about effect modification by season or temperature was performed in other studies conducted in other countries and some results were consistent with our results. A previous time-series study conducted in Shanghai, China reported that the daily counts of coronary heart disease (CHD) morbidity in the cold season (November-April) were higher than those in the warm season (May-October) [36]. They also found a significant association between the particulate matter concentration, including PM2.5 and PM10 at lag 01 and CHD outpatient and emergency department visits in all seasons. Although these significant particulate matter effects were observed in all seasons and cold seasons, the association was not statistically significant in the warm season. Research conducted in Hong Kong also found detrimental effects of air pollution in cool and dry seasons. In cool and dry seasons, a 10 µg/m 3 increment of lag 03 exposure was associated with an increase in emergency IHD admissions. However, another study conducted in Belgium showed a steep linear association in summer, whereas in winter, the association was non-linear [37]. Studies conducted in U.S. cities also showed that for the 10 µg/m 3 increase in 2-day averaged PM2.5, the percent increases in all mortality categories were greatest in the spring [38].
For the strengths of our study, although many studies have analyzed the association between PM2.5 concentration and heart diseases, such as in the UK, US and other developed countries, those PM2.5 exposure levels are quite low [39,40]. Therefore, it is important to identify the relationship between PM2.5 exposure and heart disease in a highly polluted region. Our average PM2.5 level during the study period was 25.7 µg/m 3 , which is higher than the World Health Organizations' Air Quality Guidelines (10 µg/m 3 , annual mean), USA National Ambient Air Quality Standards (12 µg/m 3 , annual mean) and Korean airquality standards (15 µg/m 3 , annual mean) [25,41,42]. We conducted the study in Seoul, a moderately polluted area with PM2.5, and found effects at higher levels of exposure. Another strength of our study is that we tried to avoid overestimating the effect of PM2.5 by constructing the co-pollutant models, and therefore we constructed both single pollutant and co-pollutant models to consider the potential role of other gaseous air pollutants such as CO, SO 2 , and NO 2 . Finally, our study is a large-scale, population-based study used KNHID data. We also used KCD codes to identify the AMI patients, which increased AMI diagnosis accuracy as it was confirmed by a physician.
For the limitations of this study, since our PM2.5 data from the Seoul Research Institute of Public Health and Environment were available only in Seoul, from 1 January 2005, we could not extend the analysis to before 2005, or to other locations except Seoul. Secondly, the air pollution level of the day was estimated according to the average of all monitoring sites in Seoul, Korea. Therefore, the possible diversity of the exposure level of each patient such as in the working area, indoor or outdoor daily activity, or the distance from the monitoring station to the patients' homes were not considered in the exposure estimation.
In conclusion, our results have demonstrated that the PM2.5 concentration was significantly associated with an increased AMI morbidity at lag 0 in Seoul, Korea. This increased association was also observed at low temperatures, suggesting that the temperature could modify the effect of air pollution on cardiovascular outcomes. Further studies from other countries that have different temperature trends are needed to examine the effect modification of PM and AMI association by temperatures. Moreover, considering the increasingly aging population, future studies identifying the effect of air pollution and temperature exposure on cardiovascular events in elderly adults are needed.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijerph18094822/s1, Table S1: Mean and the variance of daily AMI counts in our study, Table S2: Average air-pollutant concentration and weather conditions during the study period (2005 to 2014), Table S3: Newly diagnosed AMI patients in Korea from 2005 to 2014. Table S4: Adjustive relative risk for daily cases of AMI events (95% CI) per 1 • C increase in temperature, Table S5: Temperature level of Seoul in each month in our study, Table S6: Adjusted relative risk for daily cases of AMI event (95% CI) per 10 µg/m 3 increase in PM2.5 when temperature levels are between 3.70-14.30 • C, in single and co-pollutant models. Informed Consent Statement: This study did not require informed consent statement.
Data Availability Statement:
Restrictions apply to the availability of these data. | 2021-05-04T22:05:39.029Z | 2021-04-02T00:00:00.000 | {
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119318513 | pes2o/s2orc | v3-fos-license | Extreme(ly) mean(ingful): Sequential formation of a quality group
The present paper studies the limiting behavior of the average score of a sequentially selected group of items or individuals, the underlying distribution of which, $F$, belongs to the Gumbel domain of attraction of extreme value distributions. This class contains the Normal, Lognormal, Gamma, Weibull and many other distributions. The selection rules are the"better than average"($\beta=1$) and the"$\beta$-better than average"rule, defined as follows. After the first item is selected, another item is admitted into the group if and only if its score is greater than $\beta$ times the average score of those already selected. Denote by $\bar{Y}_k$ the average of the $k$ first selected items, and by $T_k$ the time it takes to amass them. Some of the key results obtained are: under mild conditions, for the better than average rule, $\bar{Y}_k$ less a suitable chosen function of $\log k$ converges almost surely to a finite random variable. When $1-F(x)=e^{-[x^{\alpha}+h(x)]}$, $\alpha>0$ and $h(x)/x^{\alpha}\stackrel{x\rightarrow \infty}{\longrightarrow}0$, then $T_k$ is of approximate order $k^2$. When $\beta>1$, the asymptotic results for $\bar{Y}_k$ are of a completely different order of magnitude. Interestingly, for a class of distributions, $T_k$, suitably normalized, asymptotically approaches 1, almost surely for relatively small $\beta\ge1$, in probability for moderate sized $\beta$ and in distribution when $\beta$ is large.
1. Introduction and summary. Individuals are observed sequentially. The problem of whether to accept an individual at the time that she is observed has a rich literature. The most celebrated version is the "Secretary Problem," where the criterion is to select one individual and the objective is to maximize the probability that the best individual is chosen. This setting has been extended in various ways including selecting a limited number for some x 0 , where α > 0. This class includes the Gamma and Normal distributions as particular cases.
The "expected overshoot," f (x) = E(X − x|X > x), plays an essential role. Our main findings are: for the "better than average" rule (β = 1), under some mild conditions, the quantity Y k − G −1 (log k) converges a.s. to a finite random variable where G(x) = x x 0 1/f (u) du. These mild conditions are satisfied in particular by the stretch exponential distributions with α ≥ 1. It is easy to show that the functions G(x) and H(x) are close to each other in that G(x) = (1 + o(1))H(x). The convergence of Y k − G −1 (log k) is shown in Section 3, where also the convergence of the sequence of expected values and variances of {Y k − G −1 (log k)} is established. The behavior of Y k for β > 1 is very different. In Section 4 we show, under mild conditions, that for β > 1, Y k /k β−1 converges a.s. to a finite positive random variable.
The behavior of T k is discussed in Section 5. It is shown that for stretch exponential random variables with α > 0, β = 1 and every ε > 0 one has T k /k 2−ε → ∞ a.s. as k → ∞, while T k /k 2+ε → 0 a.s. When α = 1, T k /k 2 converges to a finite positive random variable. The "standardized" variable for β ≥ 1 is considered and has a very interesting behavior for the stretch exponential with α > 0. For different values of β we obtain different asymptotic behavior: we show that for 1 ≤ β < 1 + 1/2α the random variable T * k converges to 1 a.s. For 1 + 1/2α ≤ β < 1 + 1/α it converges to 1 in probability. For β > 1 + 1/α the random variable T * k converges in distribution to an exponential mean one distribution, while for β = 1 + 1/α the convergence in distribution is to a sum of conditionally independent exponential random variables. We conclude with Section 6, which contains further comments and remarks. Section 2 contains some preliminaries. Proofs are relegated to the Appendix in order to highlight the results in the paper.
2. Mathematical preliminaries. The observations are denoted by X 1 , X 2 , . . . and are i.i.d. random variables from a common absolutely continuous distribution F . We assume that 1 − F (x) > 0 for all x < ∞, unless stated otherwise.
The behavior of rules will be characterized by considering two quantities: • T k = The number of observations inspected until the kth item is retained (including that item). • Y k = The average score of the first k items that are retained.
The β better than average rule is defined as follows: for fixed β (which is suppressed in the notation) and T k defined above as the number of items observed until the kth item is selected, let T 1 = 1 and Y 1 = X 1 . Define T k and Y k inductively by T k+1 = inf{i > T k : X i > βY k }, k = 1, 2, . . . , It is clear that Y k increases in k for β = 1. If β > 1 we assume nonnegative X i to avoid the situation that if Y k is negative then the cutoff to retain an observation becomes less stringent.
2.1. Theorems on almost sure convergence. In this subsection, first we present two theorems, that exist in the literature, which will be useful in proving asymptotic results for the quantities of interest. First, we shall need the following result, due to Robbins and Siegmund [6], quoted as follows: Theorem 2.1. Let (Ω, F, P ) be a probability space and F 1 ⊂ F 2 ⊂ · · · a sequence of sub-σ-algebras of F . For each n = 1, 2, . . . , let z n , β n , ξ n and ζ n be nonnegative F n -measurable random variables such that E(z n |F n−1 ) ≤ z n−1 (1 + β n−1 ) + ξ n−1 − ζ n−1 .
Then lim n→∞ z n exists and is finite and ∞ n=1 ζ n < ∞.
Proof. This follows trivially from Theorem 2.1.
We also need the following theorem that appears in Feller [2], page 239.
Classes of distributions. Preater [5] showed that when F is exponential and β = 1, Y k − log k converges almost surely to a Gumbel distribution. Krieger, Pollak and Samuel-Cahn [4] extended this result in several ways. The asymptotic behavior of other quantities, such as T k , were obtained, values of β > 1 were considered and other F , such as the Pareto and Beta, were analyzed.
An interesting question is how the rules behave for other distributions F . This depends on the behavior of the overshoot, X − a|X > a, and its expectation f (a), Note that Definition 2.1 is identical to definition (1.3) in Section 1.1 of [7], except that we refer to the auxiliary function by f A instead of f to distinguish it from the expected overshoot function. To link the two functions, we define g(u) = f (u)/f A (u). The representation of a VM distribution function F in (2.2) is equivalent to It is shown in [7] that a twice differentiable VM distribution satisfies It can be shown that lim uրx F g(u) = 1 follows from (2.4).
Let G(x) be defined by .
It is clear by this definition that H(x) = (1 + o(1))G(x) as x ր x F . We shall consider only such VM for which x F = ∞. (But see Remark 3.1.) Some of our results that will follow hold for VM distributions, but most of the results pertain to a rich subclass. Specifically: This class of distributions is denoted by G α . By change of variables y = c 1/α x it suffices in the sequel to consider only c = 1.
The reason for extending the stretched exponential by adding h(x) is to include many of the classical families of distributions such as Normal, Gamma, Lognormal and Weibull. For example, the right-hand tail probability of the standard normal behaves like φ(x)/x by Mills' ratio where φ(x) is the standard normal density. Hence the standard normal belongs to G 2 with h(x) = log(x).
3. Average, when β = 1. In this section we consider the behavior of Y k , the average after k items are retained, using the better than average rule. The emphasis is on the random variables that are generated from a VM distribution. In the first subsection, we consider the almost sure behavior, and in the ensuing subsection, results for the expectation and variance of Y k are presented.
The results are based on the following relationship: The results depend on the expected overshoot f (a) = E[Z(a)]. We shall use the following lemma later, that gives the expected overshoot and squared overshoot for F in G α for large values of a. Specifically: The proof of the results uses l'Hôpital's rule on E(Z(a)) = ∞ 0 (1 − F Z(a) (y)) dy for the expected overshoot and E(Z 2 (a)) = 2 In some instances we need a more refined result on the rate, that is the o(1) term, which depends on h(x). An easy case, as shown in the proof of Corollary 3.1, is when h(x) = 0, in which case the rate of o(1) is 1/a α .
In the more general case, we want to include h(x). The point of adding h(x) is to extend our results to known distributions such as the Normal. The role that h(x) plays, is that it is small relative to x α .
The following lemma provides a handle on the overshoot.
These conditions on h(x) and its derivatives are hardly restrictive as the intent is for h(x) to be small. In particular, if h(x) is x γ for γ < α, then all of the above conditions hold.
The details of the proofs throughout this and the remaining sections of the paper appear in the Appendix.
3.1. Results on almost sure convergence of the mean. The main result in this subsection is that under mild conditions Y k − G −1 (log k) converges almost surely to a finite random variable (Theorem 3.2). This is an extension of the result in [5] that Y k − log k converges a.s. to a Gumbel distribution when observations are generated from an exponential distribution. Theorem 3.1, which is simpler than Theorem 3.2, considers only the G α class of distributions. This theorem standardizes Y k by dividing it by a function of k. Theorem 3.2, however, provides a stronger result, which for the G α class of distributions is applicable when α > 1.
The following theorem requires a slight strengthening of condition (2.9).
1 is used to show that S k converges almost surely. We do not believe that the strengthening of condition (2.9) by (3.5) is necessary for the conclusion to hold, though we use it in the proof. We know from Theorem 3.2 that it is not needed for α > 1.
The second result of this subsection, Theorem 3.2, is the stronger statement that Y k − G −1 (log k) converges a.s. to a finite random variable as k → ∞. The conditions for this result are different from those of Theorem 3.1, but distributions in G α with α > 1 satisfy the conditions of Theorem 3.2 without the additional conditions on h made in Theorem 3.1.
Theorem 3.2. Let β = 1 and F be a VM distribution. Then under conditions: (A) EZ 2 (a) < a γ for some 0 < γ < ∞ and all a > a 0 and converges a.s. to a finite random variable as k → ∞.
The core of the proof is to show that [Y k − G −1 (log k)] 2 converges almost surely, using Theorem 2.1.
Conditions (A) and (B) are usually satisfied for F a VM distribution when G(x) increases fast enough. In particular they hold for F ∈ G α with α > 1. That condition (A) holds (for all α > 0) follows from Lemma 3.1. Condition αx α−1 ]} −1 , so from (2.9) f (x) is eventually decreasing. The case α = 1 holds when h(x) is increasing. If F has increasing failure rate (IFR), that is, satisfies "new better than used," then condition (B) is satisfied.
converges a.s. to a finite random variable as k → ∞.
Remark 3.2. The conclusion of Theorem 3.2 does not hold for all F ∈ G α , α > 0, thus not for all VM, for example, H(x) = x 1/2 . We omit the proof.
3.2.
Results on convergence of moments.
4. Average, when β > 1. In this section we consider the behavior of Y k under the more stringent condition that an observation is retained only if it exceeds β times the previous average, where β > 1. The main result is that Y k must be standardized by an entirely different quantity, namely, k β−1 , in order to get a.s. convergence. For F ∈ G α this standardization is correct for all α > 0. The result depends on the following relationship: The result concerns B k = Y k k β−1 . Let F k denote the σ-field generated by Y 1 , . . . , Y k . It follows by dividing both sides of (4.1) by k β−1 that Hence if the expected overshoot is bounded, it follows from Theorem 2.1 that B k converges almost surely. A more refined result appears in the next subsection followed by remarks on special cases. The section ends with results showing that under some conditions the expected value and variance of B k also converge. Almost sure convergence of the mean. We first show that B k converges almost surely under more general conditions in the following: Also, under the more restrictive condition of bounded expected overshoot that was used to introduce this section (which led to an easy proof of almost sure convergence of the desired quantity), (4.3) holds.
The following is a general statement about convergence of Y k for the stretched exponential family of distributions.
There exist VM distributions for which B k fails to converge a.s. to a finite limit. The proposition below provides a general result for when B k does not converge to a finite limit a.s.
If there exists a constant a 0 and a nonnegative random variable V , not identically zero, such that for all a ≥ a 0 V is stochastically smaller than Z * (a), then B k → ∞ a.s. as k → ∞.
, which is easily seen to be a VM distribution. Let Ψ(a) = a/ log(a). We shall show that the conditions (and hence the conclusions) of Proposition 4.1 hold for this example.
Convergence of moments.
We now turn to showing that the expectation of Y k suitably normalized converges to a finite limit for all random variables that belong to the stretch exponential.
We first consider EB k and Var B k in a general setting. (c) EZ 2 (a) < ca for some c > 0 and a > a 0 ; EB k and Var B k converge to a finite limit.
The above theorem does not apply for F ∈ G α , with α ≤ 1. Nevertheless, EB k converges in this case as shown in the following theorem.
Proof. By Theorem 4.2 we need only consider the case α ≤ 1. From Lemma 3.1 it follows that for some c and k large enough Substituting this into (4.2) yields Taking expectations on both sides of (4.5) and using Corollary 2.1 yields the result.
5. Time until k items are kept.
5.1.
Discussion of the problem. In this section we turn to the second quantity of interest, T k , the number of items that are observed until k items are retained. Unfortunately, it is generally impossible to normalize T k by a function of k and achieve almost sure convergence to a nondegenerate random variable. Instead we consider the following quantity: which depends on the averages {Y j }, the expectation of which tends to 1.
The results are obtained for the G α , α > 0 class of distributions. One interesting facet of the results for α ≥ 1 is that the nature of the convergence depends on β. When β is relatively small, 1 ≤ β < 1 + 1 2α , then the convergence is almost sure to 1. When β is moderate in size, 1 + 1 2α ≤ β < 1 + 1 α , the convergence is to 1, in probability. Finally, if β is large, β ≥ 1 + 1 α , the convergence is in distribution to an exponential or a sum of conditionally independent exponential random variables with means summing up to 1.
The proof uses Theorem 2.2 by conditioning on the responses {Y k }, letting with T 0 = 0. Though Theorem 5.1 gives no explicit order of magnitude of the convergence of T k , in terms of k, we get an idea of this magnitude in the following: For the exponential distribution T k k 2 converges a.s. to a limit as shown in [4].
5.3.
Asymptotic results when β > 1. The focus is on T * k , the number of observations that are observed until k items are retained suitably normalized as defined in (5.1).
For the sake of clarity, we consider in the continuation only The result for β = 1 + 1 α is of a different nature, and hence is treated separately in Theorem 5.3. To prove parts (ii) and (iii) of Theorem 5.2, we compute the limiting generating function of T k , suitably standardized, and are able to recognize the distribution for which this limit is the generating function. The results then follow from the Continuity theorem. This line of reasoning is also used in proving Theorem 5.3.
Note that for U ∼ Geo(p), . We ignore the first observation which adds one to T k (this will have no effect on the limiting distribution). Hence the resulting random part of T k (which we refer to asT k ) is the sum of, conditionally on where the sequence γ(k) is positive, and will be defined as a function of the given {Y k }, according to the need in the proof for each particular instance, but always tends to 0. Thus For (ii) we let γ(k) = 1/ k−1 j=1 e (βY j ) α and for (iii) we let γ(k) = e −(βY k−1 ) α . We now turn to the case where β = 1 + 1/α, so that β − 1 = 1/α. This is the only case where conditioning on the sequence {Y k } plays a role in the limiting distribution obtained. We know from Theorem 4.1 that there exists a random variable W , 0 < W < ∞, such that Y k /k 1/α k→∞ −→ W a.s. Our result will be stated in terms of the value of W .
where, conditionally on W = w, the R j are independently, exponentially distributed with mean µ j , where Note that the µ j sum to 1.
6. Concluding remarks. The present paper extends the results in [4] where the Exponential, Beta and Pareto distributions are considered in detail, to other distributions that include the Normal, Gamma and Weibull. The results on the special distributions considered in [4] are "invertible" in the sense that rates of convergence for Y k and T k imply rates of convergence for the number of items that are kept and the average of the items kept after n items are observed. The results obtained for the distributions considered here are in general not invertible in this way.
Preater in [5] considered the behavior of the average of the first k items that are kept, Y k , when the distribution generating the observations is exponential and β = 1 in the β better than average rule. He observed that Y k − log k converges a.s. and in L 2 to a Gumbel distribution. The behavior of this quantity for β > 1 is markedly different. When β = 1, Y k − log k converges a.s. When β > 1, Y k /k β−1 converges a.s. In addition, the rate when β > 1 holds for many distributions, while the amount that one subtracts from Y k when β = 1 depends on the distribution.
There are two interesting mathematical observations. First, it is not surprising that there should be some relationship between the domain of attraction to which the extremal distribution of F belongs and the limiting distribution of Y k , since the Y k process will, on the average, select larger and larger items. Preater in [5] shows that Y k − log k and max{X 1 , . . . , X k } − log k have the exact same limiting Gumbel distribution when the observations are i.i.d. from an exponential distribution (though Y k converges a.s. and in L 2 while the maximum converges only in distribution). Will the limiting distribution of Y k , and M k = max{X 1 , . . . , X k } always agree, or at least have the same rate of convergence? From the general theory of extreme values it follows that This should be compared with our result for β = 1 (under the appropriate conditions of Theorem 3.2), The "normalization" is the same in (6.1) and (6.2) if and only if f (x) ≡ 1, that is, if and only if the tail of the distribution of X is exponential.
The second interesting mathematical observation is that for the Beta and Pareto distributions, discussed in [4], we get the same kind of a.s. convergence for T k , after normalization (depending on β) for all β ≥ 1. In the families of distributions considered in the present paper, different kinds of asymptotic convergence hold for different values of β. Specifically, when β is relatively small, the normalized quantity converges almost surely. When β is in the middle range, the convergence is in probability. For large values of β the convergence is in distribution. So Note that H ′′ (x)/(H ′ (x)) 2 tends to 0 for a VM distribution. Now to get the rate, consider H(x) = x α + h(x) where lim x→∞ h ′ (x)/x α−1 = 0 by (2.9), and Since the first term is 1/H ′ (a), using l'Hôpital's rule on the second term yields Finally, to get the rate at which f (a)/(a 1−α /α) goes to 1, we need the rate at which h ′ (x)/x α−1 goes to 0. If we assume that h ′ (x)/(x α−ε−1 ) goes to 0, where 0 < ε < α, we have (3.4).
Proof of Theorem 3.1. Let
Hence, Taking conditional expectations on both sides, using (2.1), we therefore get . The first two terms in (A.2) therefore cause no problem in the application of Theorem 2.1 to S k . By Lemma 3.1, for all k sufficiently large so the second term in the last expression is summable, and again factoring out S k−1 the first term is also summable.
It remains to deal with the last term in (A.2). From (3.4), Thus the first term in the square brackets in (A.2) satisfies where ω = ε/α. The last line in (A.2) can therefore be rewritten as We want to study when (A.3) is positive for large k. This depends on the term in brackets, which to simplify notation we denote by R(x), where x = A k−1 , and the dependence of R(x) on k is implicit. Note that for k sufficiently large O(log k/k) < δ k ≡ 1/(log k) ω . Also note that The aim is to show that (A.3) is positive only when 1 − cδ k ≤ x ≤ 1 + cδ k for a suitably chosen constant 0 < c < ∞. We consider two cases: , the values of x such that R(x) < 0, or, equivalently, the values of x such that x α R(x) < 0 include the values of x such that R(x) < 0. It suffices to consider The set of x such that (A.6) holds is equivalent to the set of x such that , the values of x such that R(x) > 0, or, equivalently, the values of x such that x α R(x) > 0 include the values of x such that R(x) > 0. Hence, we want to consider when Since δ k and ν k are arbitrarily small for k sufficiently large, there exists x 0 > 0 such that for (A.7) to hold it is sufficient that x > x 0 . Therefore, (A.7) is equivalent to for k sufficiently large for a suitable chosen constant 0 < c 2 < ∞.
The above analysis shows that (A.3) can be bounded from above by zero when A k−1 is outside the interval c ± δ k . When it is inside, (A. Proof of Theorem 3.2. The first step in the proof is to show that (Y k − G −1 (log k)) 2 converges a.s. to a finite random variable as k → ∞.
Since Y k → ∞, there will be a (possibly random) k 0 such that for all k > k 0 , everything written below holds. Consider k > k 0 only. Let c k = G −1 (log k). Then, by (2.7) and the boundedness of f , Note that the last equality in (A.9) follows since f is bounded by condition (B). Now write Taking conditional expectation, conditional on F k−1 , yields .
We shall show that the conditions for Theorem 2.1 hold. We shall examine each term in (A.11) separately. We first show that for any ω > 0 since f is bounded (where we have denoted its bound by B). It follows that W k (ω) converges a.s. to a finite limit, L(ω) ≥ 0. Then also W k (ω/2) → L(ω/2) a.s. But W k (ω) = W k (ω/2)/k ω/2 , thus the limit must be 0 for all ω.
Term (iii) is summable by (A.9) and the boundedness of f . Term (iv) is negative, and hence causes no problem. Term (v): note first that by (A.9) where d k is a value between Y k−1 and G −1 (u k ). Since G −1 is increasing, it follows from (A.10) that The first term on the right-hand side of (A.16) can be combined with (i) in (A.11), and the second is clearly summable. Then It follows that in all cases we can write where B k , D k and V k are nonnegative random variables, and B k and D k are summable. Thus by Theorem 2.1 s. It remains to show that when W = 0, Y k − c k cannot jump between √ W and − √ W an infinite number of times. It will then follow that the limit exists and is either and that by (A.9) 0 < c k − c k−1 < γ k , for some γ > 0. Take expectations on both sides of the inequality in (A.13). Then
Thus by the Borel-Cantelli lemma
W an infinite number of times, that is, Y − c k will converge a.s. to √ W or − √ W . Since for W > 0, there always exists a small enough ε > 0 such that W − ε > 0, it follows that Y k − c k converges. Clearly on the set where {W = 0} the statement Proof of Corollary 3.1. The expected overshoot given X > a is The right-hand side follows by change of variables to y = x α . But in Abramowitz and Stegun [1], page 263, This implies that as a → ∞.
Proof of Theorem 3.3. Upon taking expectations on both sides of (A.11) we obtain .
All we need to do is subtract E[(Y k−1 − c k−1 ) 2 ] on both sides and sum. If a term remaining on the right-hand side is positive then we need to show that it is summable. If a term is negative it must be summable as the term on the left-hand side is nonnegative. Hence we see that terms (ii), (iii) and (iv) cause no trouble. The only term of concern is (v). But the expectation (integral over the density of Y k ) can be divided into an integral over three regions: As in the proof of Theorem 3.2, the integrand for regions (i) and (ii) is negative and over the third region it is positive, but can be dealt with in the same way as in the proof of Theorem 3.2 by use of Corollary 2.1. The last two statements of the theorem follow.
Proof of (i).
) which implies that B k converges, to a finite or infinite limit.
Suppose first that the limit is infinite. Then there exist k 0 and D > 1/β such that for all k > k 0 , B k−1 > D. But then from (i), for k > k 0 But the term multiplying B k−1 on the right is summable, which implies that (A.21) satisfies the condition of Theorem 2.1, and hence B k converges to a finite limit. This contradiction implies that B k converges to a finite r.v. a.s.
Proof of (ii). Now suppose that B k converges a.s. and lim EB k < ∞. It follows from (4.1) and as in (A.20) that B k can be written as where O(1/k 2 ) is positive. It follows that B k > B k−1 , so that the limit is positive. Since the support of the observations is not bounded, in a different realization one could obtain a higher value. Hence the limit is a nondegenerate positive random variable. Set B 0 = 0. Then Since B k converges a.s. lim B k exists and is finite a.s. Taking expectations and limits as k → ∞ on both sides of (A.22) and noting that EB k is Since B k converges a.s. to a random variable W β for 0 < ε < W β and a (random) k 0 , we have for all where the inequality follows since ( k−1 k ) β > ( 1 2 ) β , and where we have written Finally, By change of variable to y = Ax β−1 the integral on the right-hand side becomes A conditional on Y k−1 once βY k−1 ≥ a 0 (which will happen with probability 1).
It follows that one can imbed the sequence Y 1 , Y 2 , . . . in a probability space so that for a constant a 1 that is independent of Y 1 and k, Hence (for k such that βY k−1 ≥ a 0 and a 1 a constant) since Ψ(a) increases in a Finally, condition on Y 1 and denote By (4.4) and what we showed above, lim n→∞ n k=1 c k = ∞. It is a straightforward application of Kolmogorov's three-series theorem (cf. Feller [2], page 317) that is not summable. This and (A.24) imply that lim k→∞ B k = ∞ a.s.
Proof of Theorem 4.2. EB k converges to a finite limit by (4.2) since f is bounded by assumption (b) We shall treat each of the three terms in (A.29) separately.
(i) It is easily seen (by taking log) that the value in the square bracket is 1 + O( 1 k 2 ). (ii) From condition (c) and the convergence of EB k to a finite limit Thus the second term in the right-hand side of (A.29) is summable.
(iii) We now show that the third term in the right-hand side of (A.29) is negative or 0: where the last inequality follows from (b). It follows that (A.29) satisfies the condition in Corollary 2.1 with z n = Var B n , and the result follows.
Proof of Theorem 5.1.
Proof. Consider the event A = {Y k /k β−1 → W, 0 < W < ∞}. We know by Theorem 4.1 that P (A) = 1, and hence we shall assume that A occurs. Let A k = {Z k > γY k−1 /k δ }. We shall show that ∞ k=1 P (A k ) < ∞ so that the result will follow from the Borel-Cantelli lemma.
where βY k−1 ≤ Q k ≤ (β + γ k δ )Y k−1 . Write, by (2.9), where |o k | < ε for k ≥ k 0 with large enough k 0 and ε > 0 arbitrary. Note that by (A.26) k 0 can be chosen to depend on Y 1 only. For ε small enough this implies The next to last inequality follows from (A.26). Hence P (A k |Y 1 ) ≤ exp{−dY 1 × k β−1−δ } for k ≥ k 0 = k 0 (Y 1 ). If δ < β − 1, ∞ k=1 P (A k |Y 1 ) < ∞, so, by the Borel-Cantelli lemma, conditional on Since when R is exponentially distributed with mean µ, log E(e −tR ) = −log(1 + µt), it is sufficient to show that the right-hand side of (A.40) converges, as k → ∞, to − ∞ j=1 log[1 + tµ j ]. First consider γ(k)e dY α k−j = 1 S j,k + T j,k , Note that Y i = Z i + βY i−1 where Z i is the amount above βY i−1 for the ith item that is kept. Hence, Fix m. Then the first limit on the right-hand side is clearly zero since Y k−j → ∞ as k → ∞. Consider the second term lim sup Equation (A.44) implies that each term in the sum of the first expression on the right-hand side converges to log(1+ tµ j ) as k → ∞. We need to show that lim k→∞ k−1 j=n log[1 + t(1 + o k (1))γ(k)e dY α k−j ] can be made arbitrarily small by choosing n to be sufficiently large (all terms in the sum are positive). Note that γ(k)e dY α k−j < 1 S j,k . For any ε > 0 choose n large enough so that Hence, γ(k)e dY k−j < e dw α (1−ε) − 1 e dw α j(1−ε) − 1 < e −dw α (j−1)(1−ε) .
Choose k large enough so that o k (1) < ε. Then Since the right-hand side goes to zero as n → ∞ the second term in the sum in (A.45) can be made arbitrarily small by choosing n sufficiently large. | 2010-11-15T09:18:43.000Z | 2010-11-15T00:00:00.000 | {
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263674623 | pes2o/s2orc | v3-fos-license | Development and usability testing of a fully immersive VR simulation for REBOA training
Background Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a potentially life-saving procedure for bleeding trauma patients. Being a rare and complex procedure performed in extreme situations, repetitive training of REBOA teams is critical. Evidence-based guidelines on how to train REBOA are missing, although simulation-based training has been shown to be effective but can be costly and complex. We aimed to determine the feasibility and acceptance of REBOA training using a fully immersive virtual reality (VR) REBOA simulation, as well as assess the confidence in conducting the REBOA procedure before and after the training. Methods Prospective feasibility pilot study of prehospital emergency physicians and paramedics in Bern, Switzerland, from November 2020 until March 2021. Baseline characteristics of trainees, prior training and experience in REBOA and with VR, variables of media use (usability: system usability scale, immersion/presence: Slater-Usoh-Steed, workload: NASA-TLX, user satisfaction: USEQ) as well as confidence prior and after VR training were accessed. Results REBOA training in VR was found to be feasible without relevant VR-specific side-effects. Usability (SUS median 77.5, IQR 71.3–85) and sense of presence and immersion (Slater-Usoh-Steed median 4.8, IQR 3.8–5.5) were good, the workload without under-nor overstraining (NASA-TLX median 39, IQR 32.8–50.2) and user satisfaction high (USEQ median 26, IQR 23–29). Confidence of trainees in conducting REBOA increased significantly after training (p < 0.001). Conclusions Procedural training of the REBOA procedure in immersive virtual reality is possible with a good acceptance and high usability. REBOA VR training can be an important part of a training curriculum, with the virtual reality-specific advantages of a time- and instructor-independent learning. Supplementary Information The online version contains supplementary material available at 10.1186/s12245-023-00545-6.
Background
Trauma is the leading cause of death in patients under 45 years of age [1].In particular, non-compressible torso hemorrhage resulting in hemorrhagic shock bears a high mortality and morbidity [2][3][4].Resuscitative endovascular balloon occlusion of the aorta (REBOA) has recently gained popularity as a potentially life-saving procedure, by allowing quick transitory hemorrhage control for truncal injuries [5][6][7][8][9].A recent meta-analysis suggests a positive effect of REBOA in non-compressible torso injuries when compared to resuscitative thoracotomy [10], as does a recent propensity score-matched analysis [11].Different international guidelines on polytrauma management suggest its application in unstable trauma patients who are unresponsive to other resuscitative efforts [12][13][14].
The application of REBOA is also under investigation during cardiopulmonary resuscitation, as it increases coronary and cerebral perfusion pressure [15,16].
However, because REBOA is a rare and complex procedure performed in extreme situations with the potential to cause great harm to the patient, mastery of this particular skill is critical.High-volume deployment centers show increased survival of REBOA patients when compared to low-utilization centers [17].
REBOA training is usually done by simulation or a combination of simulation with knowledge transfer done with lectures or/and eLearning.Although a recent systematic review confirms the effectiveness of simulationbased training, there is still confusion about optimal course design, effect size, skill transfer, and skill retention [18], and evidence-based guidelines on how to train REBOA are missing.Simulation training is usually done with a high-fidelity endovascular simulator (e.g., Mentice VIST, Mentice, Gothenburg, Sweden) or live animal models [18].Traditional simulation-based training therefore is very resource intensive, including high costs for training materials (e.g., high-fidelity manikin, REBOA training catheters) and personnel resources.
Virtual reality (VR) is a technology that immerses the user in an artificial 3D environment with the use of a head-mounted device (VR headset).Interaction with the virtual environment takes place via wearable devices (controllers) or even with the user's own hand (hand tracking).VR simulations have proven to be a useful and effective tool, mainly for training surgical and technical skills [19][20][21][22].VR simulation training offers a scalable, autonomous (time-and location-independent) experience, especially for settings that prove to be too risky or resource-intense for traditional simulations.
To our knowledge, there is no fully immersive virtual reality simulation for REBOA training so far.
At the "Schutz und Rettung Bern" [23], we recently started a clinical trial on REBOA in non-traumatic cardiac out-of-hospital cardiac arrest [24].While the actual insertion of the REBOA catheter in this setting is performed by a core team of 4-5 senior emergency physicians, we need to train the regular prehospital teams (prehospital emergency physicians and emergency paramedics) in the basic principles of the REBOA procedure so that they understand the procedure and can assist the core REBOA team.We thus aimed to.
i) Develop a fully immersive VR REBOA training simulation
ii) Determine the feasibility of the application of the VR REBOA simulation at the local prehospital emergency medical services iii) Evaluate the acceptance of the VR REBOA simulation (usability, simulator sickness, sense of presence and immersion, workload, user satisfaction) iv) Examine the subjective confidence in conducting the REBOA procedure before and after the simulation training.
Development of the fully immersive VR simulation
To realistically recreate a medical procedure in VR, we have to understand its basic steps, actions, and milestones.The best way to obtain such a breakdown analysis is to consult professionals specialized in this method.In our case, the Subject Matter Experts material, required for the design of a complete storyboard tailored for the REBOA VR training module, was provided by our medical experts (TB, WEH, TCS) to the development team of ORamaVR (Geneva, Switzerland), according to the methodology published in [25].
The simulated environment consisted of an emergency theatre including a virtual patient, who is hemodynamically unstable after a motor vehicle accident with free fluid in the abdomen.Clinical information, vital signs, ultrasound, or X-ray images, as well as information on the next steps are displayed on monitors in the virtual emergency room.A sterile covered table is used to store and prepare the required materials.The insertion of the REBOA catheter is performed step by step on a virtual person.These steps include.
1. Decision on placement depth and measurement for zone 1 REBOA using the clinical information 2. Preparation of femoral arterial access 3. Cannulation of the common femoral artery using ultrasound 4. Guidewire introduction 5. Placement of sheath 6. REBOA catheter preparation 7. REBOA catheter insertion 8. Balloon inflation and confirmation of its effect 9. Fixation of catheter 10.Chest X-ray Screenshots of the simulation are detailed in Figs. 1 and 2. Two modes of action were available for single-player use: In the tutorial mode visual aids and prompts helped the trainees in providing information on the next procedural step; these prompts were missing in the normal mode.This study was exempt from full ethical review by the local institutional review board (Kantonale Ethikkommission Bern (Ethics Committee Bern), BASEC-No: Req-2020-00970).Written informed consent for study participation was obtained from all participants.Written informed consent from a parent and/or legal guardian is "not applicable".Consequently, the present study
Participants
The local prehospital emergency medical services ("Schutz und Rettung Bern") are carrying out about 23,000 preclinical medical emergency operations annually.There are 17 emergency physicians and 108 paramedics (50% female) working in a rendezvous system.All participants were offered and attended the training on a voluntary basis and we provided no remuneration.Written informed consent was obtained for the study and publication of the study results.Written informed consent from a parent and/or legal guardian is "not applicable".
Baseline data
Sociodemographic data (gender, age, profession (physician/paramedic), working experience in years, need to wear eyeglasses, right/left-handedness), prior training and experience in REBOA as well as prior experience with VR, were collected in a survey.
Intervention
Initially, three peer teachers were introduced to the VR set-up and the correct operation of the REBOA VR module by the development and study team of the University Emergency Department (TS, TB, JB) in a 2-h training session, who then passed on their knowledge and were able to train their peers ("teach the teacher").The REBOA VR simulation station was set up in an empty room at the Schutz und Rettung headquarter Bern (Fig. 3).The hardware consisted of a stand-alone VR headset with two hand-held controllers (Oculus Quest, Oculus VR, Facebook Inc., Menlo Park, CA, USA) and a tablet pc.The REBOA module, version 1.2.6, software platform, developed by ORamaVR (Geneva, Switzerland), was used in the single-player tutorial and normal mode.
During 5 months, from November 2020 to March 2021, paramedics and preclinical emergency physicians had the opportunity to train with the VR simulation on their own after being instructed by the trained peer instructors during their shifts or whenever was a suitable timeslot for them.The peer instructors had the opportunity to follow the simulation on the tablet PC, and thus to provide additional targeted assistance, apart from the automated feedback by the tutorial mode of the software, if necessary.
Outcome measures Acceptance of the VR simulation
Evaluation of acceptance of the VR simulation was carried out according to established questionnaires directly after the VR training.
Usability
Usability was assessed using the System Usability Scale (SUS) [26], which is composed of 10 questions with a 5-point Likert attitude scale and the After-Scenario Questionnaire (ASQ) [27], which assesses the ease of task completion, satisfaction with completion time and
Sense of presence and immersion
Presence and immersion in the virtual world were determined according to the 6-item questionnaire developed by Slater-Usoh-Steed (total score ranges from 1 = no immersion to 7 = full immersion) [29].
Workload
Perceived subjective workload on a scale from 0 to 100 was assessed using the NASA-Task Load Index (NASA-TLX) as a total score and within 6 subdomains [30].Overstraining is associated with a total score > 60, and understraining with a total score of < 37 [31].
User satisfaction
The User Satisfaction Evaluation Questionnaire (USEQ) has six questions with a 5-point Likert scale to evaluate user satisfaction (total score ranges from 6 = poor satisfaction to 30 = excellent satisfaction) [32].
Furthermore, free-text comments were collected.
Subjective effectiveness/confidence
Confidence in the correct performance of the REBOA intervention was assessed before and after the training ("I feel confident in conducting the REBOA intervention correctly" (Likert scale from 1 = totally disagree to 5 = totally agree).
Statistical analysis
Data was analyzed using SPSS.Baseline characteristics are presented as numbers and percentage or median and interquartile range (IQR) using descriptive statistics as appropriate.Pre-and postsimulation comparisons were performed with the Wilcoxon signed rank test.A p value < 0.05 was considered significant.
Development of the VR REBOA simulation
To create the VR training, we used MAGES 4.0, which enables rapid prototyping of shared, collaborative networked medical training in VR [32].
Feasibility
VR REBOA training for paramedics and emergency physicians was found to be feasible.The chosen peer teaching format was well accepted and implemented by the participants and confirmed as a very useful approach.The use of the VR simulation in only one empty room without further equipment was possible and enabled spontaneous practice sessions without scheduled training hours or permanently reserved training rooms and personnel (Fig. 3).
Baseline characteristics of the study population
Baseline characteristics of the study population are detailed in Table 1.Of the 45 participants, 4 (8.9%) were physicians.None of the participants had received prior training in REBOA or had ever carried out the procedure in real life.Participating physicians had limited experience in femoral arterial cannulation.The majority of the participants did not regularly use of video games or VR.Free-text comments of the participants generally indicated a good acceptance (e.g., "Great way to practice courses of action or scenarios"; "I thought the VR experience was great and was able to get a good look into the REBOA catheter procedure").However, critical aspects were illuminated as well (e.g."Interesting experience to have a VR headset on my head for once.But for me this is no substitute for other means of education, as there is too much support needed.Cost/benefit ratio is not right for me.").The complete free-text comments of the participants are detailed in Supplement 1.
Confidence
Subjective confidence of the participants in using the REBOA procedure correctly before training was low and significantly increased after the VR simulation (Table 3).
Summary
Training with a fully immersive VR simulation for REBOA is feasible with a good usability, high satisfaction, and optimal workload during training.We showed that the VR training increased familiarity with the procedure with little VR training-associated side effects.
Feasibility
Our study shows that it is possible to set up and conduct REBOA VR training on a population without prior knowledge of the procedure and in any given location without specific preparations necessary.Compared to a traditional simulation center, this type of implementation does not require any special constructional prerequisites but only an empty playing area.
The study participants found their way easily within the simulation and were able to run through it independently after a brief introduction, although they had no previous experience with VR or other computer games.Since the simulation has a tutorial mode and a realistic game character, the use was easy and intuitive for the majority of the test subjects.Some participants initially needed help from a peer teacher outside of the simulation, which could be provided through observation on an adjunct tablet computer.Since the VR training is an autonomous gaming experience, it does not necessarily require a trained instructor with specific medical knowledge.The peer instructor concept is based on the "teach the teacher" principle and can thus save expensive human resources as well as instructor time.As the system shows and constantly controls the correct execution and sequence of the skills to be learned, the VR simulation can be used time and instructor-independent.
Acceptance
The usability, measured with the System Usability Scale (SUS), was high.To evaluate user satisfaction, the key component of usability from different perspectives, we confirmed good user satisfaction with both ASQ and USEQ.When evaluating a new training method such as VR, it must be taken into account that the evaluation may be subject to the novelty effect, and the usability measurement results may be overestimated [33].
In addition to high satisfaction and good usability in general, participants indicated they experienced a high level of presence and immersion in the VR training without significant side effects.This high level of immersion and presence could be achieved through immersive VR technology with head-mounted displays, which, to the best of our knowledge, we are investigating for the first time for a REBOA training, and by avoiding immersioninterrupting elements, e.g., dialog boxes or drop-down menus whenever possible.It has been argued that a high level of presence and immersion in VR can be an indicator of cognitive engagement with the content of the virtual environment, and thus an important predictor of experiential learning [34,35].
Confidence
Although the confidence after the VR training had increased significantly in the pre-post-comparison, a relevant number of participants still reported a low confidence.
Since VR training is limited in terms of haptic experience and knowledge transfer is also easier to teach using classic learning methods, such as self-study e-learning, it is recommended to integrate a VR training into a dedicated learning curriculum.VR training is not intended to replace any other training but to supplement it.An example is the out-of-hospital-cardiac-arrest training curriculum by Brede et al. [36].However, this problem is not specific to our REBOA training setting shown here but has already been demonstrated in other settings.Since our goal was not to train and enable our participants to self-administer the REBOA catheter in the planned clinical trial, we simply wanted to improve their knowledge and understanding of the procedure and enable them to support the REBOA core team in the field.
Another possible application of VR training could be self-guided training to prevent skill decay, a well-known and relevant problem in the teaching of skills [37].Park et al. showed REBOA skill degradation was most pronounced in surgical trainees who did not receive training for more than 5 months [38].There are no studies to date on the best way to teach REBOA in the long term and how to minimize skill decay.However, the persistence of knowledge learned in VR over one month was previously demonstrated for procedural teaching [35].Given the time-, location-, and instructor-independent nature of our REBOA training, it could be an ideal way to support regular self-guided repetition training to prevent skill decay.Further long-term research is needed.
Limitations
These results must be interpreted with some limitations.First, this was a study of a single population with a limited number of participants who may be subject to selection bias due to voluntary participation, thus impacting generalizability.Although technically possible in the present VR simulation, in our study setting the training was conducted by only one person at a time.This lacks the opportunity to train teamwork skills that are essential for effective work in emergency settings.However, procedural skills are the basis of any teamwork and the present simulator was designed to teach these procedural basics.Likewise, a non-haptic VR simulation cannot be a substitute for haptic skills training such as sonography-guided vascular puncture, underscoring the use of VR simulation as a supplement to, rather than a substitute for, a REBOA training curriculum.
In our study, only a subjective measure of the effectiveness of the training was conducted using self-rated confidence pre-and post-training.
Due to the infrequency of the trained procedure, objective outcomes at the patient level are difficult to collect.One potential approach for future studies could be the use of the REBOA rate instrument [39].
Conclusion
The procedural training of the REBOA catheter procedure in immersive virtual reality is possible with a good acceptance and high usability indicated by the trainees.REBOA VR training can be an important part of a training curriculum, with the virtual reality-specific advantages of a time-and instructor-independent learning.
This is a prospective feasibility pilot study involving prehospital emergency physicians and emergency paramedics of the "Schutz und Rettung Bern" [23].The study was conducted at the University Emergency Department (Universitätsklinik für Notfallmedizin) at the Inselspital, University Hospital, Bern, Switzerland.The study took place from November 2020 until March 2021.
Fig. 1 Fig. 2
Fig. 1 Screenshot of the VR REBOA simulation.Preparation of the sheath, adaptable view into the torso
Fig. 3
Fig. 3 Setup of the VR simulation.Setup for the VR REBOA simulation including hand-held controllers, head-mounted device (Oculus Quest), tablet PC, disinfection materials Abbreviations: ASQ After-Scenario Questionnaire, IQR interquartile range, NASA-TLX NASA-Task Load Index, SUS System Usability Scale, USEQ User Satisfaction Evaluation Questionnaire
Table 1
Baseline characteristics of the study population (n = 45) Abbreviations: IQR interquartile range, no number, REBOA resuscitative endovascular balloon occlusion of the aorta, VR virtual reality
Table 2
Acceptance of the VR REBOA simulation
Table 3 Confidence
Abbreviation: REBOA resuscitative endovascular balloon occlusion of the aorta | 2023-10-06T13:53:36.836Z | 2023-10-06T00:00:00.000 | {
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246703864 | pes2o/s2orc | v3-fos-license | Association of the Serum Folate and Total Calcium and Magnesium Levels Before Ovarian Stimulation With Outcomes of Fresh In Vitro Fertilization Cycles in Normogonadotropic Women
Background Women of reproductive age are recommended to consume folic acid and other supplements before conception and during pregnancy. We aimed to investigate the association of the serum folate and total magnesium (Mg) and calcium (Ca) levels before ovarian stimulation with the outcomes of assisted reproductive technology (ART) in normogonadotropic women. Methods We used a subanalysis of data obtained from a multicentre, randomized prospective study (NCT03088137). A total of 110 normogonadotropic, non-advanced aged, non-obese women with tubal and/or male infertility factors were enrolled for the single fresh ovarian stimulation GnRH antagonist cycle. The main outcome measures were the total oocyte yield, mature oocytes, fertilization rate, biochemical, clinical pregnancy, and live birth. Multivariable generalized linear models adjusted for covariates were used with a Poisson distribution and the log link function for adjusted oocyte counts, and a binomial distribution and the log link function were used for adjusted clinical ART outcomes. Results The medians (interquartile range (IQR)) were as follows: baseline serum folate, 20.55 ng/ml (10.8, 32.9); Mg, 19.4 mg/L (18.7, 20.7); Ca, 94 mg/L (91.2, 96.4); and Ca/Mg ratio, 4.78 (4.55, 5.02). Women with higher serum folate concentrations (Q4≥33.0 ng/ml) had significantly lower total numbers of oocytes retrieved (adjusted mean (95% CI) 9.2 (7.6-11.3) vs 12.9 (10.9-15.4, p-trend=0.006)) and lower odds ratios (ORs) (95% CI) of 0.12 (0.02, 0.79) for clinical pregnancy and 0.10 (0.01, 0.70) for live birth compared with women in the lowest quartile (<10.8 ng/ml), all p-trend<0.001. Women in the highest Ca/Mg ratio quartile (≥5.02) had ORs (95% CI) of 6.58 (1.31, 33.04) for biochemical pregnancy, 4.85 (1.02, 23.08) for clinical pregnancy and 4.07 (0.83, 19.9) for the live birth rate compared with women in the lowest quartile (<4.55), all p-trend<0.001. Conclusions Using multivariable models, we suggested that a baseline elevated serum folate level (≥33.0 ng/ml) and a lower Ca/Mg ratio were associated with worse ART outcomes in normogonadotropic women. Our findings might be useful for choosing safe dosages of folate, calcium, magnesium and complex supplementation for both fertile women and women undergoing infertility treatment. Further preconception large-scale studies with known micro- and macronutrient statuses of both parents and serum folate, Ca, Mg, and hormone levels, are needed.
INTRODUCTION
Micronutrient deficiencies before and during pregnancy affect reproductive health and increase the risks of adverse pregnancy outcomes for mothers and children. The International Federation of Gynaecology and Obstetrics (FIGO) recommends a varied and healthy diet and provision of supplements or fortified foods throughout pregnancy and the periconceptional period aiming to compensate for possible deficiencies and increased nutrient requirements (1). For most women who are planning to become pregnant, a wide range of over-the-counter complex vitamins, mineral preparations, amino acids and botanicals are recommended for use before and during pregnancy (1-3); however, personal requirements based on individual levels in body and diet characteristics are not considered. Overconsumption of micronutrients can affect pregnancy outcomes and children's health, including an increased risk of early fetal death (4) and altered psychomotor development (5).
Folic acid or folate (natural form) is an essential micronutrient for many physiological processes, and its nutritional demands increase during pregnancy (1,3,6). The recommended dosage of folic acid from supplementation in addition to dietary folate intake is 400 mg/day and is based on the prevention of neural tube defects (1,3,6). However, more than 57% of women did not reach the recommended dosage of 400 mg/day during pregnancy, 25% took more than 1000 mg/day and 3.5% consumed >5000 mg/day of folic acid supplements (5).
Calcium (Ca) and magnesium (Mg) are other important micronutrients for pregnant women (3). A high incidence of Mg deficiency was demonstrated in a Russian observational study among pregnant women and women with hormonedependent diseases (7,8). It was shown that reductions in serum levels of calcium and magnesium during pregnancy might be possible contributors to the etiology of preeclampsia and that supplementation of these elements to the diet may be of value to prevent preeclampsia (9). In other studies, hypocalcemia correlated with severe preeclampsia, while magnesium supplementation did not reduce the incidence of preeclampsia in pregnant women (10)(11)(12). Muneyvirci-Delale et al. reported the recurring cycling of ionized Mg and cyclic alterations in the ionized Ca to Mg ratio in the serum of healthy women over the menstrual cycle, which was accompanied by changes in the levels of the sex steroids, estradiol, progesterone and testosterone (13).
Adequate folate status has also been broadly investigated before and during assisted reproductive technology (ART), and significant differences in the prevalence of folic acid intake among countries have been revealed (14)(15)(16). The results of studies investigating the folate status, supplementation before and during ART and ART outcomes are rather controversial. On the one hand, a higher serum concentration of folate before ART treatment was associated with higher live birth rates among American women using different treatment protocols (17). Conversely, women with unexplained infertility had significantly higher median plasma folate concentrations than fertile women, and a good folate status did not have a positive effect on pregnancy outcome following ovarian stimulation (OS) (18). Moreover, higher concentrations of serum folate can modulate the ovarian response to gonadotropin during OS with GnRH-agonist by influencing estradiol levels, which is associated with a lower estradiol response and lower follicle number in the folic acid users group in comparison with nonusers (19). It was shown that a diet high in synthetic folate can be associated with increased endogenous progesterone levels and a lower risk of sporadic anovulation (20).
Using our data obtained from a therapeutic equivalence study of follitropin alpha biosimilar (21), we aimed to investigate the associations of serum folate, vitamin B12, magnesium and calcium levels before ovarian stimulation with the outcomes of ART in a single GnRH antagonist (GnRH-ant) protocol in normogonadotropic women.
MATERIALS AND METHODS
This was a subanalysis of data obtained from a multicentre, randomized, embryologist-blinded, parallel-group, therapeutic equivalence study of follitropin alpha biosimilar (ClinicalTrials.gov Identifier: NCT03088137) (21). The major results of this study argue in favour of both investigated follitropins being therapeutically equivalent; therefore, the clinical data obtained in this study for randomized patients in both compared groups can be combined and considered for further analysis in the current substudy.
Ethics Approval and Consent to Participate
The study protocol and informed consent were approved by the Russian Ministry of Health (RCT 754 dated 26.10.16)
Study Population
The flowchart of study recruitment and follow-up is presented in Figure 1. In 2017-2018, 114 women were enrolled in the parental study of the therapeutic equivalence of follitropin alpha in three IVF centres in Moscow, Russia ("AltraVita" Human Reproduction Clinic, Perinatal Medical Center, and Lapino Clinical Hospital) using the following inclusion criteria: women aged 20-35 with a regular menstrual cycle; tubal and/or male causes of infertility factors; first or second attempt at IVF/ ICSI; 18 ≤ BMI ≤ 30 kg/m 2 ; FSH level <10 IU/l, estradiol level <50 pg/ml and AMH level ≥1.0 ng/ml at cycle days 2-5; 4 ≤ antral follicular count (AFC) ≤ 15; no pathology of the endometrium; and no sexually transmitted infections (21). Three women were excluded due to contradictions to ART (n=2) and a severe male factor (sperm concentration <2 10 6 /ml, n=1), and 1 woman refused. A total of 110 women were enrolled and followed up within the ART protocol prior to outcomes by 14 obstetricians who had standardization training before the study. The serum of all 110 women from parental study NCT03088137 was analyzed for micronutrients.
Clinical Procedures
No tight restrictions for folic acid or other food supplements/ vitamins were applied before, during or after OS, with the usual recommendation of taking 800 mg/day folic acid. Participants underwent a single OS follicular-phase GnRH antagonist protocol in one fresh cycle. Starting on days 2-3 of the menstrual cycle after the screening period, a fixed daily subcutaneous (SC) dose of 150 IU of follitropin alpha (Gonalf ® , Merck, Germany or Primapur ® , IVFarma, Russia) was administered daily for 5 days, with possible correction after day 5 based on ultrasound examinations ( Figure 2) (21). A GnRH-ant ganirelix acetate (Orgalutran ® , MSD, Netherlands) at 0.25 mg was added daily starting when the leading follicle reached a mean diameter of 14 mm less than 37 hours after the intramuscular administration of either 5000-10 000 IU of hCG (Pregnyl ® , MSD, Netherlands), n=92 (84%), or 0.2 mg of GnRH-a (GnRH agonist) (Decapeptyl ® , Ferring Pharmaceuticals, Switzerland) in 18 (16%) women at risk for ovarian hyperstimulation syndrome (OHSS). The hCG trigger criteria were leading 2-3 follicles ˃ 18 mm; the GnRH-a criteria in women at risk of OHSS were growth of more than 15 follicles ˃ 14 mm on the day of trigger. Transvaginal oocyte retrieval was performed, followed by IVF or ICSI according to the centre's standard procedures as well as luteal phase support. The transfer of a maximum of 2 embryos/blastocysts was allowed after oocyte retrieval with endometrial thicknesses not less than 8 mm.
Biospecimen Collection and Assessment of Micronutrients and Hormones in the Serum
Blood samples were collected at 2-5 days of the menstrual cycle during the screening period within 4 weeks prior to day 1 and the administration of follitropin alpha ( Figure 2). Fresh serum and whole blood were transported to three accredited clinical laboratories, INVITRO, CITILAB, and the laboratory of the Perinatal Medical Center, MD Medical Group, Moscow, Russia, for assessments of the blood count, biochemical parameters, including total protein, parameters of hemostasis and the following hormones: AMH; FSH, LH, estradiol, prolactin, total testosterone, cortisol, TSH, ACTH, and GH. Serum for micronutrient analysis [folate, B12, total magnesium (Mg) and calcium (Ca)] was stored below -70°C (22) and analyzed in one batch at "Chromolab", Moscow, Russia. The serum folic acid and vitamin B12 levels were measured by an Architect i2000 microparticle chemiluminescent immunoassay analyser (Abbott, USA) with a LOD of 0.5 ng/ml and a LOQ of 1.5 ng/ml for folate and 125 pg/ml and 150 pg/ml for vitamin B12, respectively. Nine samples of the 110 (8%) were above the upper limit of detection for folate (49 ng/ml) and were treated as 49 ng/ml. The serum total Ca and total Mg concentrations were measured by atomic absorption spectrometry (AAS) using an AA-7000 spectrometer (Shimadzu, Japan) with a LOD of 3 mg/l and a LOQ of 5 mg/l for Ca and 1 mg/l and 4 mg/l for Mg, respectively. All samples were within the detection limits. The calcium/magnesium ratio (Ca/Mg ratio) was calculated using values measured in mg/l.
Covariate Assessment
Fourteen trained obstetricians collected covariate information, including age, height, weight, calculated BMI, history of infertility (duration and causes, IVF/ICSI attempts), and reproductive and applied clinical parameters. The diet was not assessed before the ART cycle. Ten embryologists evaluated semen quality of male partners and embryo quality routinely in accordance with their own standards implemented in each clinic. Given no specific standardization training before the study for embryologists to assess semen and embryo quality and expected high between-observer variability, we did not use parameters of basic semen analysis as covariates and grades of embryo quality as outcomes in main models.
Outcome Assessment
Seven consecutive outcomes of ART efficiency were evaluated. Early ART outcomes for 110 women included the number of follicles ≥ 16 mm on the day of hCG (or GnRH-a) administration; the number of oocytes retrieved; oocyte quality outcome (MII stage of maturity); and the number of fertilized oocytes (zygotes with two pronuclei, 2PN). Fertilization was not successful (the number of fertilized oocytes was zero) for one participant of 110, and for two, there was no embryo to transfer. For 7 women with a high risk of OHSS, embryo transfer was cancelled. Therefore, for those 10 women for whom embryos were not transferred for reasons potentially related to predictors (no embryo to transfer, high risk of OHSS), their subsequent outcomes were considered negative. Two participants dropped out of the study due to movement from clinics for family reasons and were excluded from the evaluation of late outcomes. Thus, the late clinical ART outcomes were estimated for 108 women and included biochemical pregnancy (on days 12-17 after embryo transfer with a positive test for beta hCG ≥ 25 mIU/ ml), clinical pregnancy (defined as a gestational sac with fetal heart activity occurring at the 10 th week after embryo transfer), and the take-home baby rate as the final outcome ( Figure 1).
Statistical Methods
Spearman correlation analysis was used for the initial search of associations between the baseline levels of micronutrients and study outcomes. Since we found that the baseline B12 level was not associated with any ART outcomes, we did not focus on B12 in further analysis. Then, women were classified into quartiles Descriptive statistics were calculated for demographic, reproductive, and laboratory characteristics according to these quartiles. Differences between the highest and lowest quartiles of investigated micronutrients were tested using the Kruskal-Wallis test for continuous variables and the chi-square test for categorical variables. Generalized linear models were used to evaluate the association between serum folate concentrations, Ca/Mg ratios and ART outcomes while accounting for withinperson correlations in outcomes. A Poisson distribution and loglink function were specified for oocyte counts, and a binomial distribution and log-link function were specified for late clinical outcomes as recommended for observational studies of ART (23). Tests for trends across quartiles were conducted by using the nptrend command by STATA (24), and individual predicted values in each model were grouped by quartiles. Confounding was evaluated by using prior knowledge and descriptive statistics from our cohort. The core model includes more than 30 covariates that can have an effect on the ART cycle. For each outcome, we fit a full multivariable model including all covariates as univariate p ≤ 0.20 for that outcome. The following covariates were considered for inclusion in models as factors: age (continuous), BMI (continuous), female and male factors of infertility (yes or no), infertility duration (continuous), number of previous ART attempts (0 or 1), type of follitropin alpha (Gonal-f ® or Primapur ® ), ovulation trigger (GnRH-a or hCG), duration of stimulation (continuous), FSH dose (continuous) and baseline serum concentrations of hormones (LH, FSH, AMH, estradiol, prolactin, and testosterone; continuous). Parameters of hemostasis (antithrombin and activated partial thromboplastin time (APTT); continuous) (25) were included, as well as red blood cell counts (RBC; continuous) because low RBC counts may indicate vitamin B12 and folate deficiency (3); and total protein level (continuous). The model for each outcome was reduced to a final model retaining covariates with p < 0.10. In these models, we included quartiles of the serum folate concentration and Ca/Mg ratio and evaluated the associations between the quartiles of the serum folate concentration, Ca/Mg ratio and each outcome. Since two oocyte outcomes, number of follicles ≥16 mm and retrieved oocytes, were highly correlated, we used only latest one for models. We conducted statistical analyses using Statistica version 12.0 (StatSoft, Inc., USA) and considered 2-sided significance levels of p<0.05 to be statistically significant. For oocyte counts, the results are presented as population marginal means adjusted for covariates. For clinical outcomes (yes or no), the results are presented as ORs and 95% CIs from a comparison of quartiles 2, 3, and 4 with quartile 1 or as predicted probability (0 or 1) adjusted for covariates.
In sensitivity analyses, we additionally adjusted models for the baseline estradiol level due to possible confounding effects, and for total protein level due to protein-bound calcium in blood.
Baseline Characteristics
The baseline demographic, reproductive, hormonal and clinical characteristics are shown in Table 1 (mean ± SD) for a total of 110 women who underwent OS and were grouped by the quartile ranges of the serum folate level and the Ca/Mg ratio. Women aged 30.7±2.8 years were enrolled in the study, and the mean duration of infertility due to tubal (35.5%), male (43.6%), and tubal and male (20.9%) factors was 41.6 ± 28.2 months. In 66.4% of cases, patients had their first IVF/ICSI attempt, while in 33.6%, patients had their second attempt. Basal levels of hormones were as follows: FSH, 6.61 ±1.87 IU/l; LH, 5.24±3.71 IU/l; AMH, 5.02±3.43 ng/ml; and estradiol, 34.85±12.54 pg/ml. The antral follicle count was 11.28 ±2.87. Together, these baseline characteristics prove the inclusion criteria for nonadvanced aged, nonobese, normogonadotropic women with tubal and/or male infertility factors and a prognostic normal response to OS that was estimated to be approximately 70% Figure 3. Two women in our study had deficient serum folate concentrations, as defined by WHO criteria, below 4 ng/ml (28).
Comparison of Baseline Characteristics in Groups of Micronutrients
Baseline demographic, reproductive and hormonal characteristics were generally similar across quartiles of the serum folate concentration and the Ca/Mg ratio. Factors considered as inclusion criteria, including age, BMI, infertility cause, baseline FSH and AMH level, were not different, while the serum estradiol level was different. Women in the lowest quartile of serum folate had significantly higher estradiol levels (37.8 ± 10.9 pg/ml) than women in the highest quartile (30.2 ± 14.1 pg/ml), p=0.046, and women in the lowest quartile of the Ca/Mg ratio had significantly lower estradiol levels (30.8 ± 13.7 pg/ml) than women in the highest quartile (38.1 ± 10.7 pg/ml), р=0.02 ( Table 1). Male partners in the lowest quartile of serum folate
Efficacy of the ART Protocol
In total, 110 women were treated (mean±SD) for 9.74±1.05 days with an average total dose of r-hFSH equal to 1525±260 IU (Table 1 and Figure 2). After fertilization, 2 subjects dropped out from the study due to movement from clinics for family reasons. For 10 patients, embryo transfers were not performed due to the high risk of OHSS for 7 women, no embryos to transfer for 2 women, and no fertilized oocytes for 1 woman. Fresh embryo transfer on days 3 (n=20, 18.2%) and/or 5 (n=78; 70.9%) was performed in 98 women of 110, and the mean number of transferred embryos was 1.27 ± 0.45.
The efficacy of the ART protocol was comparable with data obtained from other trials with different FSH regimens, as summarized in meta-analyses (29, 30) ( Table 2). The mean number of follicles ≥ 16 mm on the day of trigger administration was 11.7 ± 5.6, the number of oocytes retrieved was 11.9 ± 6.8, the number of matured oocytes (MII stage) was 9.8 ± 5.9, and the number of fertilized oocytes (twopronuclear zygote, 2PN) was 8.7 ± 6.0. A total of 35.7% of women had biochemical pregnancies, 29.6% had clinical pregnancies, and the take-home baby rate was 27.6%.
Collinearity of Covariates (Inclusion Criteria) and Micronutrients
Using Spearman correlation, we checked the potential collinearity and association of investigated serum micronutrients and covariates, particularly factors that were considered inclusion criteria. Age, the baseline serum AMH level and the number of antral follicles were not correlated with micronutrient levels. BMI and the baseline serum FSH level were positively correlated with the baseline calcium level, with rho=0.24, p=0.01, and rho=0.28, p=0.003, respectively. The serum estradiol concentration was significantly positively correlated with the Ca/Mg ratio (rho=0.27, p=0.005) and significantly negatively correlated with the Mg level (rho=-0.22, p=0.03), while the negative correlation of the baseline estradiol level with the folate level (rho=-0.15, p=0.11) was not significant. Total protein level was significantly positively correlated with both, serum calcium and magnesium level (rho=0.29, p=0.004 and rho=0.24, p=0.019, respectively), but not with Ca/Mg ratio (rho=-0.10, p=0.31).
Covariates and ART Outcomes
Based on univariate regression analysis, age, male factor of infertility, sperm concentration, baseline levels of total protein, estradiol and LH, type of follitropin alpha, ovulation trigger, and
(29.6)
Take-home baby rate, n (%)* 27 (27.6) *no data about the sex of new-borns. day and number embryo transfer were not associated with biochemical and clinical pregnancies or the live birth rate. A lower age, a higher baseline level of estradiol and the use of GnRH-a as an ovulation trigger (vs hCG) were associated with higher numbers of retrieved, matured and fertilized oocytes, all with p<0.05. Other covariates significantly associated with outcomes that were included in the final multivariate models are presented further.
Baseline Ca/Mg Ratio and ART Outcomes
The calcium and magnesium levels (expressed as Ca/Mg ratio) did not have statistically significant impacts on early ART outcomes, including the oocyte yield (p-trend=0.88), number of matured oocytes (p-trend=0.16) and fertilized oocytes (p-trend=0.15), with the same adjustments as for serum folate ( Table 3 and Supplementary Figures S1A-C).
Women with a higher Ca/Mg ratio had significantly higher biochemical (p-trend=0.000), clinical pregnancy (p-trend=0.000) and live birth rates (p-trend=0.000) ( Table 4 and Figures 6A-C). Specifically, women in the highest quartile of the serum Ca/Mg ratio (≥5.02) had a 6.58 (95% CI: 1.31, 33.04) odds ratio (OR) for biochemical pregnancy ( Figure 6A); an OR of 4.85 (95% CI: 1.02, 23.08) for clinical pregnancy ( Figure 6B); and an OR of Analyses were conducted by using generalized linear/nonlinear models with a Poisson distribution for oocyte counts and the log link function for all outcomes with adjustment for continuous BMI, baseline serum AMH and APTT, duration of stimulation and dose of FSH. c Tests for trends across quartiles were conducted by using the nptrend command by STATA (24), and individual predicted values in each model were grouped by quartiles.
4.07 (95% CI: 0.83, 19.9) for live birth ( Figure 6C), with the same adjustments as for serum folate, compared with women in the lowest quartile (<4.55) ( Table 4). Women in the 3 rd quartile of the Ca/Mg ratio (4.78-5.01) had a significant OR of 7.09 (95% CI: 1.45, 34.6) for biochemical pregnancy and a tendency of a higher OR for clinical pregnancy (3.14 (95% CI: 0.66, 14.9)) and life birth (2.65 (95% CI: 0.54, 12.9)). There was no change in the association between the Ca/Mg ratio and ART outcomes when adjusting for the baseline serum estradiol level. There was no change in the regression models when adjusting for total protein.
DISCUSSION
Adequate vitamin and micronutrient levels are important for oocyte quality, maturation, fertilization, and pregnancy outcomes. However, the amount of biological data accumulated today cannot provide straightforward clinical recommendations for each vitamin and biologically active compound with a focus on human reproduction and ART outcomes (1,2). In the present work, we investigated the impacts of the baseline serum folate and total Ca and Mg levels (expressed as the Ca/Mg ratio) on ART outcomes in a single fresh GnRH-ant treatment cycle. A total of 110 normal responder women without endocrine and ovarian disturbances (normogonadotropic nonadvanced aged, nonobese with a tubal and/or male infertility factor) were enrolled in the study. They represent one of the main clusters of infertility: fallopian tube blockage or damage occurs in more than 20% of women referred to fertility clinics (31,32), and up to 40% of cases involve a male factor (33). According to recommendations for normal responder patients (34,35), the single GnRH-ant protocol using the starting fixed 150 IU daily dose of r-hFSH followed by its adjustment on the basis of the ovarian response was used in this study.
In this study, we found that among nonobese, normogonadotropic women aged 20-35 years, women with higher serum folate levels (≥ 33.0 ng/ml) had significantly lower total numbers of oocytes retrieved and lower odds ratios of 0.24 (0.04, 1.37) for biochemical pregnancy, 0.12 (0.02, 0.79) for clinical pregnancy and 0.10 (0.01, 0.70) for live birth compared with women in the lowest quartile (<10.8 ng/ml).
Recommendations for folate/folic acid intake differ between countries, although the majority of guidelines recommend a healthy diet plus folic acid supplementation at 400 mg/day from the preconception period (up to 3 months) until the end of the first trimester of pregnancy, as issued by the WHO (6). Based on WHO recommendations that first appeared in 1968 addressing the macrocytic anemia concern and that are still active, a deficiency of folate status can be considered with serum folate levels below 3 ng/ml; possible deficiency with levels of 3.0-5.9 ng/ml; the normal range is 6.0-20.0 ng/ml; and "elevated" levels are more than 20 ng/ml (27). According to WHO criteria, the majority of women in the present study (n=83) had normal (n=28, quartile Q2 in the study (10.8-20.5 ng/ml)) and "elevated" (n=55, quartiles Q3 and Q4) serum folate levels, and women with possible deficiency and normal folate levels were included in quartile Q1 (<10.8 ng/ml, Tables 1, 3, 4).
To date, many studies have tried to assess both the effects of folate intake deficiency and the risk of excessive folic acid intake among women in the periconception period. Inadequate folate intake first leads to a decrease in the serum folate concentration, followed by a decrease in the erythrocyte folate concentration, an increase in the homocysteine concentration, megaloblastic anemia, and changes in the bone marrow and other tissues with rapidly dividing cells (28,36). In addition, there is strong evidence that folate deficiency during and before pregnancy is associated with low birthweight and neural tube defects (6). Conversely, some studies revealed adverse effects of higher doses or longer consumption of folic acid. Cheng et al, 2019 reported that a longer duration of folic acid supplementation before pregnancy might increase the risk of gestational diabetes mellitus (37). High dosages of folic acid supplements during pregnancy can influence psychomotor development after the first year of life (5). Recently, Field and Stover, 2018, reviewed the safety of folic acid and suggested that additional research is needed to assess the health effects of folic acid supplement use when the current upper limit for folic acid is exceeded (38). The median (IQR) B12 level was 350 (239, 471) pg/ml in our study, which is higher than the 200 pg/ml considered inadequate by the WHO (28) and higher than the 300 pg/ml recommended for women becoming pregnant to reduce the risk for neural tube defects (39). However, serum B12 concentrations in our study were lower than those measured in women in the EARTH study by Gaskins et al, 2015, who reported positive associations of the serum concentrations of vitamin B12 and folate greater than the median (534 pg/ml for B12 and 20.5 ng/ml for folate) with a higher live birth rate (17). In our study, the baseline vitamin B12 concentration was not associated with any ART outcomes. We did not find folate and B12 correlation (rho=0.15). Furthermore, we did not observe high serum folate concentrations or coexisting B12 deficiency ( Table 1), which can lead to hyperhomocysteinemia (40).
The biological pathways involved in the action of folates and other micronutrients on the efficacy of ART are still unclear. The possible mechanism that can partially explain the results is related to the interplay between folates, magnesium/calcium levels and estradiol biosynthesis.
As shown in Table 1, women in the highest quartile of the serum folate concentration had significantly lower baseline serum estradiol levels than women in the lowest quartile, and a higher serum estradiol level was associated with a higher number of retrieved, matured and fertilized oocytes in univariate models. The steroidogenic potential of ovaries depends on aromatase enzyme complex activity, which converts androgens produced by theca cells to estrogens in granulosa cells (41). This process is modulated through different endocrine, paracrine and autocrine factors and mechanisms. Among them are insulin-like growth factors (IGFs), which affect cell growth and metabolism and are responsible for the release of estradiol from human granulosa cells in a dose-dependent manner (42). Previously, it was found that IGF-1 significantly enhanced the expression of aromatase (CYP19A1) mRNA and caused increased estradiol production (43). IGFs act by binding to specific IGF receptors (types I -II), which are also localized in granulosa cells (42). It has been shown that the IGF-I receptor gene is a novel downstream target for folic acid action (44). Namely, folic acid downregulates IGF receptor type I promoter activity as well as its endogenous mRNA expression and protein levels in a dosedependent manner (44). The lack of expression of IGF receptors can lead to diminished estradiol biosynthesis in granulosa cells. Previously, it was found that higher folate levels during the IVF programme might modify the ovarian response to OS and have an influence on estradiol levels either directly by modulating follicular FSH responsiveness or through aromatase availability (19). Additionally, a folate-rich diet could have the effect of lowering circulating IGF-1 levels in elderly women (45).
DNA methylation is a key and the most investigated component of epigenetic modifications as well as nutritional epigenomics (46). There is excessive evidence that nutrients, especially folates as methyl donors, may modify DNA methylation as a main physiological process for gene silencing that leads to biological pathway activation/deactivation (46). For example, folate, as a precursor to S-adenosylmethionine, is an essential cofactor for the methylation of catechol estrogens and other derivatives (47) that can also affect estradiol biosynthesis in granulosa. Recently, it has been reported that periconceptional folate levels at both paternal and maternal levels are associated with epigenetic events in gametes, embryos and children (48)(49)(50)(51). Another important and new finding of our study was that a lower baseline serum Ca/Mg ratio was associated with worse clinical ART outcomes in a dose-dependent manner. As shown in Table 1, women in the lowest quartile of the Ca/Mg ratio had significantly lower estradiol and calcium levels and higher magnesium levels than women in the highest quartile, while the median (IQR) serum calcium and magnesium concentrations in the present study were 94 (91.2, 96.4) mg/l and 19.4 (18.7, 20.7) mg/l, respectively, and considered normal (52). Investigating five different stages of normal cycling (the menstrual, early follicular, late follicular, ovulatory and luteal phases), Muneyyirci-Delale et al, 1998 presented physiological recurring cycling of ionized Mg and cyclic alterations in the Ca/Mg ratio in the serum of healthy women of reproductive age (13). They reported an increase in the serum Ca/Mg ratio at both the ovulatory and luteal phases when the estradiol level was increasing. The positive significant correlation of the estradiol level and the Ca/Mg ratio in our study may reflect the physiological processes in women described earlier (13).
Based on previous studies, it was shown that calcium and magnesium can modulate estrogen levels and their metabolism. For example, an overall positive association was found between circulating levels of IGF-I and serum calcium (53). Therefore, higher calcium (and a higher Ca/Mg ratio) can be associated with higher IGF-1, which is responsible for the release of estradiol from granulosa cells in a dosedependent manner (42). Moreover, magnesium is a cofactor of catechol-O-methyl transferase and influences the methylation and excretion of catechol estrogens (54,55).
We need to address the strengths and limitations of our prospective study. As strengths, aiming to minimize residual confounding, we used a single IVF protocol with fresh embryo transfer in one cycle per normogonadotropic woman. We controlled for more than 30 factors, including a history of infertility (duration and causes, IVF/ICSI attempts), reproductive and applied clinical parameters, baseline hormone levels, biochemical and hemostasis parameters and blood cell counts. Hormone laboratory and clinical staff were blinded to the micronutrient serum levels.
As a limitation, we did not stratify cohort by the male factors including semen quality and their micronutrient status that can influence ART outcomes. However, we collected information about the male factor as the cause of infertility and found that male factor was not associated with ART outcomes in univariate regression models. Additionally as a limitation, we did not estimate the dietary intake and supplement consumption, and we did not measure the serum vitamin D3, iron, and zinc, which can also affect folate, calcium and magnesium levels and ART outcomes. We did not use expensive direct measurement of ionized calcium or albumincorrection of calcium. Instead of this, we used well standardized assessment of total calcium and provided sensitive analysis using adjustment for total protein. We did not found change in association between the Ca/Mg ratio and ART outcomes when adjusting for total protein. We measured hormones in three different accredited clinical laboratories; however, micronutrients were measured in one lab by one batch. Single time-point for lab measurements, the relatively small sample size and selective inclusion criteria are other important limitations. However, "normal responder" (normogonadotropic non-advanced aged, non-obese with tubal and/or male infertility factor) women who were invited in our study were one of most common groups of patients seeking infertility treatment in IVF clinics (26).
It is worth noting that different nutrients are essential during the periconceptional period and pregnancy and can modulate hormone signalling and epigenetic programming of gametes, and it might be associated with early development and offspring health. Special attention should be given to ART cycles that are accompanied by additional epigenetic risks to improve the efficacy of cycles and to provide better health of children. ART cycles can be improved not only by state-of-the-art techniques and treatments but also by guided consumption of "well-known" biologically active substances and nutrients before, during and after ART. Further preconception large-scale studies with known micro-and macronutrient statuses of both parents, including daily doses and serum folate, Ca, Mg, homocysteine and hormone levels, are needed.
CONCLUSIONS
In multivariable models, among nonobese normogonadotropic women aged 20-35 years, a baseline higher serum folate concentration (>33.0 ng/ml) was associated with worse ART outcomes, including biochemical and clinical pregnancies as well as the live birth rate. A baseline lower Ca/Mg ratio (<4.55) was associated with worse clinical ART outcomes in a dosedependent manner. Our findings might be useful for choosing a safe dosage of folate and other supplementations for both fertile women and women undergoing infertility treatment.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.
ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Russian Ministry of Health (RCT 754 dated 26.10.16) | 2022-02-11T14:20:57.052Z | 2022-02-11T00:00:00.000 | {
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43908270 | pes2o/s2orc | v3-fos-license | A classification system for causes of occupational accidents for use in preventive strategies.
A classification system for causes of occupational accidents for use in preventive strategies. Scand J 1991;17:302-11. A comprehensive classification system, which allowed operational analysis of the events preceding accidents, was applied to the analysis of information surrounding the occurrenceof all traumaticwork-related fatalitiesin Australia in 1982-1984. The coded information included factors immediately antecedent to the accident leading to the fatality and factors removed in time which contributed to the occurrence of the accident. The complex network of events leading up to the accident, their interrelationships, and their relative contribution to causing the accident were examined. The results provide information about the use of accident analysis for the formulation of preventive strategies.
Epidemiologic descriptions of the occurrence of occupational accidents provide information about the nature , distribution, and rate of work-related injury and death (1). They describe how, when, and where accidents occur and are therefore important starting points for identifying preventive strategies. However, classifying accidents by type or severity of injury, or by agent or place of injury, provides limited information about why accidents occur. Comprehensive knowledge about the causes of occupational accidents has the potential to provide much more specific information on which to base preventive efforts.
Several frameworks have been proposed for analyzing the causes of accidents (2)(3)(4)(5)(6)(7)(8)(9). However , most of these frameworks have not been comprehensive in that they have not considered the range of possible causal factors and their interrelationships. The range of causal factors has been restricted both in the extent to which different types of factors are considered and also in the extent to which the wider circumstances surrounding the occurrence of the accident are included. In some instances, only one type of causal event, for example, human error (2, 7), or a broad general category of causal factor, for example, behavioral factors (3), has been considered in detail.
Alternatively, where a wide range of factor types has been included, it has been at a relatively superficial level. Consideration of some of the more complex causal factors, such as human error, has typically been much less detailed (5,10).
Similarly, lack of comprehensiveness and detail has also been evident in the extent to which circumstances I National Institute of Occupational Health and Safety, Sydney, Australia. Reprint requests to: Dr A-M Feyer, National Institute of Occupational Health and Safety, GPO Box 58, Sydney NSW 2001 Australia. 302 surrounding the accident are considered. Although several frameworks have considered only those events immediately precipitating the accident (8, II) , more typically, most accident analysis frameworks recognize the potential importance of both immediate precipitating causes and the wider circumstances surrounding the occurrence of the accident (5,(12)(13)(14)(15). However, analysis of the wider circumstances has often not been as detailed as for events occurring immediately before the accident (12,13).
The nature of the relationships between factors is another important dimension of the analys is of accident causes. Several studies have considered the relative sequential nature of causal factors such that they define when different factors occur in the accident sequence (5,9,12,13). The relative causal nature of relation ships between different factors in the accident sequence has not been studied. Since it is clear that all factors are not of equal causal significance, having information about the relative importance of factors in accident causation must improve the specificity with which targets for prevention can be defined (14). This information, however, is rarely gathered.
Some models of accident causation have attempted to acknow ledge the causal contribution of factors . Frameworks based on fault-tree analysis have provided the most restrictive view of the causal contribution of factors (6,9,12,16). They have sought to identify the necessary and sufficient relationships between the events in the accident sequence by use of logic rules. Other approaches have taken a more conceptual view of the relative importance of causal factors (5,14), the events immediately precipitating the accident being seen as only symptomatic of the underlying or preexisting conditions which contribute to the occurren ce of the accident. The underlying factors, located in the wider circumstances of the occurrence of the accident , are seen as causally more significant. The most parsimonious approach, adopted only rarely, has been to impose a weightingsystemin order to establish the relative importance of factors without any preconceptions about the role that either immediate or more removed factors might play (10).
Classification systems have been developed which recognize the need for the most comprehensive treatment possible of information available about the occurrence of the accident (14,16). These systems have still to be applied to large banks of accident data. In part, the lack of application reflects the fact that the frameworks involve distinctions which can be specified at a theoretical level, but are often difficult to operationalize, especially when the process is retrospective. The specification of complex logical relations between events (6,16), for example, is limited in applicability in retrospective accident analysis.
The aim of the present research was to design a comprehensive classification system for occupational accidents which would describe the array of causal factors, map their relative sequential relationships, evaluate the relative importance of factors in accident causation, and, yet, be operationally applicable. For the evaluation of its applicability, the classification system was applied to a large heterogeneous data base of occupational accidents.
Methods
The study population was derived from all workrelated traumatic deaths occurring throughout Australia in the years 1982-1984. The data were collected from coroners ' reports in a study of work-related fatalities conducted by the National Institute of Occupational Health and Safety (as described in reference 1). Of the 1738 fatalities judged to be work-related by Harrison et al (1), 1020were included in the current study. Cases were excluded on the following grounds: (i) case was not in active work at the time of the fatality, (ii) case was not of working age, that is, below 15 years or above 65 years of age, (iii) death occurred during work commuting, (iv) death due to a road traffic accident when the vehicle was not the person 's normal workplace, and (v) death occurred when person was on recess from active work or was a bystander to work. Of the remaining cases, 130were excluded becausethey did not contain enough information to be coded . The study therefore included all fatalities that occurred while the person was actively involved in work over the three-year period of study.
The coroners' reports were relativelycomprehensive. They contained a textual account of the circumstances of the accident and subsequent fatality, as well as demographic information about the person, medical information regarding the nature of the injuries and cause of death, and details of all investigations into the causes of death .
The classification system
The classification system was developed to establish, from the information in the coroners' reports, the sequence of events surrounding and leading to the fatality. It was designed to allow coding of a sequence of up to three events which immediately preceded the accident and which led to the fatality , as well as any further factor(s), classified into one of eight possible categories, that made a direct contribution to the occurrence of the accident or fatality. These were titled precursor events (abbreviated to PEl , PE2, or PE3, depending on location in the sequence with respect to the accident) and contributing factors, respectively. Not all of the accidents involved the full range of precursor events and/or contributing factors . For the purposes of coding, time before the fatality referred to the relative sequential order of the events leading up to the fatality . Conventional units of time were therefore variable. In other words, PEl was closest to the accident and PE2 occurred earlier than PEl in the accident sequence, but the time separating the events could have varied from seconds to minutes to hours or even perhaps days. Figure 1 is a schematic representation of the possible event sequence.
The nature of each of the three possible precursor events was coded into one of the following categories: (i) environmental -events resulting from the location of the accident and are events that could not have been changed at that point in time, (ii) equipment -events resulting from breakage or malfunctions that occur in machinery or tools at that point in time, (iii) medical -events resulting from the person 's current state of physical well-being, and (iv) behavioral -events resulting directly from human involvement. Each category was coded in terms of the certainty of evidence for its existence, with 1=yes, certainly; 2=yes, probably; 3 = yes, possibly; 4 = indeterminate; 5 = no.
Behavioral events were coded further in terms of who performed the behavior (the deceased or another person) and whether the behavior constituted an error (ie, the incorrect performance of standard operating procedure).
Errors were coded with the use of two well-known error classification systems to enable the assessment of the ease with which they can be used for the kind of data available in this study and their utility for preventive strategy formulation (17). They were Swain's (I 8) task-related error classification and Rasmussen's (7) classification of the functional level of behavior during which the error occurred. The results of this coding have been discussed elsewhere (17).
The nature of the contributing factors was coded into the following categories: (i) environment -factors resulting from the location of the accident occurring at an earlier time, (ii) equipment -factors associated with the design of machinery, tools, personal protective equipment or safety equipment, (iii) work practice -factors involving poor or risky standard operating procedures accepted by management and/or personnel (this included separate categories of poor upkeep or misuse of equipment, (iv) supervision -factors relating to inadequate charge of workers, (v) training -factors relating to inadequate training of workers, (vi) task error -factors relating to incorrect performance of duty, (vii) medical-factors involving physical well-beingat an earlier time, and (viii) other -factors such as alcohol/drug involvement, delays in receiving medical treatment and social aspects. The coding for contributing factors was again in terms of the certainty of evidencefor the existence of the contributing factor. One factor that was not included in the present classification system was injuring energy. All injury is most directly caused by energy release in excess of injury 304 thresholds (19). Thus energy transfer was assumed to occur in all cases in the present study, and therefore the classification of its presence or absence would have been redundant. Classification of the nature of the energy involved is highly relevant to the nature, agent, mechanism, and severity of injury. In contrast, the focus of the present classification system was on the causes of the accident leading to injury. Therefore, it was those factors that initiated and influenced the build-up of energy which were coded.
Information was also collected on demographic details, details of the person's job at the time of death, the nature of their usual job (if different), the use of personal protective or other safety equipment, and the degree of forewarning of the accident.
Ranking of causal factors
The precursor events and contributing factors leading up to the fatality were ranked in terms of their significance for the occurrence of the fatality. The chain (or chains) of events and factors were ranked such that the root cause was deemed as the most significant (scored" I "), and occurrences following directly from the root were ranked by diminishing degrees as significance decreased (ie, 2, 3, 4, etc) . The root causes were termed prime causes. An important feature of the ranking procedure was that the prime cause or causes of an accident could be located in the contributing factor categories or the precursor event sequence. Therefore, it was possible for contributing factors to be ranked as more causally influential than precursor events in any given accident. This feature of the ranking procedure allowed the separation of causal importance and temporal importance of events; events were coded both in terms of proximity to the accident and causal significance such that the two were not necessarilyequivalent. It was also possible to code more than one prime cause for any accident sequence.
The coding instrument was developed by three coders test-coding a random sample of 30 cases. The results were then discussed and the coding instrument modified to balance the scope of information collected with the quality of information available. Once the instrument was finalized, a single coder coded all cases.
An example
The following is a hypothetical example of the coding of an accident scenario into event categories and the ranking of those event categories for causal significance.
Scenario. While standing on a catwalk above large machinery, a worker leaned too closely into the path of the operating machinery. He was dragged into the machinery. The guard usually on the catwalk had been removed several days pre viously to facilitate repair of the machinery and had not been replaced. The general work area was poorly lit. Interpretation. The event immediately preceding the accident was an error on the part of the worker when he leaned too far into the path of the machinery. Operating the process without the usual safety railing allowed the error to occur, while the poor lighting in the work area made its occurrence much more likely.
Reliability
Inter-and intrarater reliability of two aspects of coding were evaluated. First, the reliability with which the same event category sequences were coded to describe the cases was evaluated. Two independent coders coded 40 randomly selected cases, and their frequency of agreement with the main coder was calculated. The main coder recoded these 40 cases after a period of six months had elapsed, and the frequency of agreement between the two coding occasions was calculated.
The second aspect of coding that was evaluated was the reliability with which the relative causal importance of the events could be ranked. There were two parts to evaluating the reliability of assigning rank to events. First, ranks were assigned to the events coded in the 40 randomly chosen cases that have already been described. In other words, where differences occurred in categorizing cases into event sequences, the coders had ranked different event category sequences for the same case. Since the event category sequences into which cases are coded critically influences the importance assigned to an event in the sequence, this was not an unambiguous evaluation of the reliability of ranking per se. Therefore, the follow ing additional method of evaluating ranking reliability was used : 40 randomly selected cases, coded into event category sequences by the main coder, were ranked by the two independent coders. In thi s procedure each coder ranked the same event category sequences. This procedure allowed an evaluation of the reliability of the ranking of events in a sequence in relation to each other, independently of coding the events into categories.
To measure the association between pairs of coders for the ranking of the events , the Guttman coefficient of monotonicity was used . A monotone trend of Y on X implies that, as X increases, any change in Y is onedirectional with the possibility that Y can occasionally remain constant (20). The coefficient varies between o and I.
Reliability
When 40 randomly selected cases were coded by two independent coders, the percentage of agreement with the main coder for coding the cases into event category sequences was 84 070 for one coder and 82 070 for the other coder. The percentage of agreement when the main coder recoded these 40 cases after a period of six months had elapsed was 89 %.
When complete cases were both coded and ranked independently, the Guttman coefficient was calculated 305 on an event-by-event basis for all possible events in the sequence (ie, for the three precursor events and the eight contributing factors) because of the possibility of different numbers of events in the sequence for different coders. The mean Guttman coefficient for one independent coder compared with the main coder was 0.68 (SD 0.25). For the other independent coder, the mean Guttman coefficient was 0.83 (SD 0.13). The Guttman coefficient for the main coder compared on the two coding occasions separated by six months was 0.89 (SD 0.09).
When coded cases were ranked, that is, the same sequences of event categories were ranked, by the two independent coders and these were compared with the rankings assigned by the main coder, the Guttman coefficient was 0.99 for both independent coders. Figure 2 shows the frequency of involvement of the major event categories and the percentage of cases in which each category was ranked as a prime causal factor. Errors, poor work practices, and environmental factors were the factors most frequently involved in accident causation. Errors showed the highest frequency of involvement, occurring in more than two-thirds of the cases. Environmental factors, either as a percursor event or as a contributing factor, were the next most common and were present in 62.4 070 of the cases.
I_ CATECORYPRESENT
Poor work practices were involved in almost half of all the cases. The remaining categories each occurred in less than 15 0J0 of the cases.
Human error at some point in the sequence was also the most common prime causal factor. In fact, almost half of all the cases involved error as a prime cause. Poor work practices were involved as a prime cause of the fatality in approximately one-third of the total number of cases. Although environmental factors were among the most frequently involved factors, they were not commonly involved as prime causal factors.
Patterns of prime causal rankings
Examination of the distribution of prime causal rankings in the event sequences revealed that prime causal factors were at least as prominent in the contributing factors as they were in the precursor sequence. In over half of all the cases (55.6 0J0) there was one prime causal factor in the precursor event sequence. Similarly, in 56.2 0J0 of the cases, there was at least one prime causal factor in a contributing factor category. Furthermore, in a little over one-third of the cases (37 OJo), prime causal factors were located exclusively among the contributing factors. In 43.8 0J0 of the cases, prime causal factors were located exclusivelyin the precursor event sequence.
Multiple prime causes occurred the most commonly as a combination of precursor events and contrib- uting factors. In 23.2070 of the cases there was at least one contributing factor with a ranking of "1" in addition to an event in the precursor sequence with a ranking of "1." In contrast, multiple prime causes were located in the precursor sequence only in 7.4 % of the cases; they were present in the contributing factors only in 10 % of the cases.
Patterns of event sequences and rankings
The precursor event sequences were examined for consistent patterns. Twelve patterns were found , each representing at least 2 % (about 20 cases) of the cases. Four of these patterns accounted for 66 % of all cases in the sample. These four major patterns are discussed in the following text. Two of these event sequences involved a behavioral event at PEL (See figure 3.) For ease of display, patterns involving other event categories at PEl were terminated at PEl and nonmajor patterns involving a behavioral event at PEl were terminated at PE2 or PE3 in figure 3. The number in parentheses under each PE label in the figure refers to the percentage of total cases represented by the sequence to that point.
Together, the two main patterns accounted for 34.5 % of all the cases, while the remaining patterns with a behavioral event at PEl accounted for only 4.1 % of the cases. In the more common of the two patterns (25.7 % of all cases), the behavioral event at PEl was the sole event in the precursor event sequence. In the other pattern, accounting for 8.8 % of the cases, the behavioral event at PEl was preceded by another behavioral event at PE2. An important distinguishing feature of these two precursor sequences relates to the relative causal importance of the behavioral event at PEL Specifically, when the pattern consisted of a sole behavioral precursor event, that event was ranked as a prime causal factor in the majority of cases (66.3 %) represented by the sequence . However, when the pattern was one in which the behavioral event at PEl was preceded by another behavioral event at PE2, it was the earlier event (at PE2) which was ranked as a prime causal factor for the majority of cases (57.3 %) represented by the sequence , rather than the event at PEl (27 % of cases). Figure 3 also shows the contributing factors associated with each precursor sequence. The number in parentheses under each contributing factor label refers to the percentage of cases represented by the particular sequence which also showed involvement of that contributing factor. The number not in parentheses refers to the percentage of cases represented by the precursor sequence for which the contributing factor was ranked as a prime cause.
Both patterns showed almost identical involvement of contributing factors. The work practice category was involved in about one-third of the cases, the environment category and the other category both being represented in approximately 20 UJo of the cases. With respect to ranking, both the work practice category and the other category were ranked as prime causal factors in more than 70 UJo of the cases in which they were present, while the environment category was ranked as a prime causal factor much less frequently (in 22 % of the cases for one pattern and in 0 % of the cases for the other).
The remain ing two major patterns were ones in which an environmental event immediately preceded the accident. (See figure 4.) Together, the two patterns accounted for about one-third of all the cases (32.1 UJo), in approximately equal proportions.
In one of the patterns, the environmental event at PEl was the sole event in the precursor event sequence. In the other pattern, the environmental event at PEl was preceded by a behavioral event at PE2. The relative causal importance of the environmental event at PEl was changed dramatically with the presence of a preceding behavioral event. When the environmental event was the sole event in the precursor sequence, it was ranked as a prime causal factor in 41.4 UJo of the cases represented by the sequence. In contrast, when the pattern was one in which it was preceded by a behavioral event at PE2, the environmental event at PEl was ranked as a prime causal factor in only 17.6 UJo of the cases represented by the sequence, while the earlier behavioral event was ranked as a prime causal factor in the majority of cases (65.5 %).
The work practice, other, and environment categories were again the main contributing factors involved. Work practice was the most common, and it was ranked the most often as a prime causal factor. Environmental factors were again less causally influential than contributing factors from either the work practice or the other category.
For the pattern in which an environmental event immediately before the accident was the onl y precursor event, there was a substantially increased involvement of behavioral contributing factors, work practice and task errors at an earlier time being the contributing factors occurring the most often . Furthermore, these contributing factors were predominantly ranked as prime causal factors.
Discussion
Inter-and intrarater reliability was high both for coding cases into event category sequences and for assigning ran kings of relative causal importance to the event categories. This finding suggests that the classification system is viable for describing the network of events preceding accidents and for evaluating their relative causal importance. The system therefore provides an important starting point for the development of a data-driven base for the formulation of preventive strategies.
Human error , poor work practices, and environmental factors were found to be the most frequent antece -307 den ts of the fatalities. However, the prim e causes of accidents were not necessarily those factors which were the most frequently involved. Error at some point in the sequence was the most frequently involved factor. It was also the mo st common prime causal factor . In contrast, environmental factors , which were involved at some point almo st as frequently as error, were prime causal factors in onl y a mino rit y of ca ses. In other word s, for most cases involving environmental factors, other factors in the accident sequence were of greater causal significance.
Nor were the prime causes of the accidents necessarily those fac tors which occurred the most closely in time to the accident. In one-third of the cases there were no prime causal factors located in the precursor event sequence. That is, the root cau se of the accident g no PEl (2.0"10 ) EmrlronmlHlta' (18.1Wo
Contlrbutln
Factors PE3 PE2 PE1 Figure 3. Major patterns of event sequences w ith a behavioral event at precursor event 1 (PEl). The number in parentheses under each PE refers to the percentage of total cases represented by the sequence to that point. For the contributing factors, t he numbe r in parentheses refers to the percentage of cases represented by the PE sequence which also showed involvement of that co ntributing fac tor, and the number tha t is not in parentheses refers to the percentage of cases represented by the sequence fo r which the contributing factor was ranked as a prime cause .
Contirbuting
Factors -~P E3 PE2 PEl Figure 4. Major patterns of event sequences with an environmental event at precursor event 1 (PE1). The number in parentheses under each PE refers to the percentage of total cases represented by the sequence to that point. For t he contributing factors, the number in parentheses refers to the percentage of cases represent ed by t he PE sequence which also showed involvement of that contributing fac tor , and the number t hat is not in parentheses refers to the percentage of cases represented by the sequence for wh ich the contributing factor was ranked as a prime cause.
2 309 for these cases was located outside the event sequence leading directly to the accident. Not only did prime causal factors occur at various locations in the accident sequence, but multiple prime causes were also relatively frequent. In about one-third of the cases, at least two prime causal factors occurred in the accident sequence, most commonly as a combination of precursor events and contributing factors .
Knowing where in the accident causation sequence a particular factor is likely to occur and how important it is likely to be provides a useful starting point for identifying targets for prevention. However, because the accidents were shown to be multicausal, more information is needed about the causal pathways whereby factors exert their influence. Without this information it is very difficult to identify where in the accident sequence, and with what sort of strategy , it may be possible to intervene the most effectively.
A striking characteristic of these results was that there were relatively few patterns of causal pathways. Indeed, the majority of the accidents were accounted for by one of four major precursor event sequences consisting of exclusively behavioral events, exclusively environmental events, or a combination of these two categories. Furthermore, similar networks of contributing factors were found to be associated with each of the four main sequences, specifically the environment, the work practice, and the other categories. The main component of the other category was found to be drug and alcohol involvement.
Factors occurring early in the sequence were identified as much more influential in causing the fatalities than events closer to the fatality. When two events occurred in the event sequence, the earlier event (PE2) was generally ranked as more important. This earlier event was a behavioral one for all patterns; therefore it was not possible to determine whether its influence was due to the nature of the event or its location in the sequence. The fact that behavioral contributing factors were always ranked highest regardless of the rest of the accident sequence suggests that a combination of these characteristics (ie, location and nature of the event) is important.
These results clearly indicate that the nature and influence of events occurring earlier in the sequence has important implications for drawing inferences about preventive strategies. Being able to identify that preexisting poor work practices and earlier task errors, and to a lessextent drug and alcohol involvement,were frequently the predisposing inputs for the events which immediately precipitated the accident provides highly specific targets for prevention.
The need for the application of comprehensive accident analysis systems to large banks of accident data has often been highlighted (3,14,21). The present study constitutes the first attempt to do so. The classification system was designed to provide the sort of information that can be used to identify targets for prevention. It comprehensively and reliably describes 310 the antecedents of the accidents, their interrelationships, and their relative causal importance. The results confirmed that accidents are the outcome of a complex network of interrelated factors which are not equivalent in causal significance. The findings also demonstrated that when information is available about the nature, temporal location with respect to the accident , and causal role of all of the factors in the antecedent network, then the targets for prevention can be much more specifically defined .
The level of analysis reported in this presentation has provided only an overviewof the major causal networks preceding this large group of fatalities. It provides some insights into the contributors to workrelated deaths in Australia. A more-detailed analysis of the involvement of human error has already been completed (17). Further analysis will focus on the differences in accident causation across industry, the detailed investigation of the nature and role of poor work practices, and the role of risk taking in accident causation. | 2018-04-03T05:43:12.984Z | 1991-10-01T00:00:00.000 | {
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4679942 | pes2o/s2orc | v3-fos-license | Malathion Neurotoxic Effects on Dopaminergic System in Mice: Role of Inflammation
Background: Pesticides exposure is considered a global health problem. Increased risk of neurodegenerative disorders has been strongly associated with chronic repeated exposure to organophosphates. The mechanism of chronic neurotoxicity is still unclear and seems to be different from cholinergic affection in acute toxicity. Methods & Findings: This work aims to verify the degenerative effects of chronic malathion exposure on dopaminergic system in BALB/c mice and to identify the possible contributing mechanism. Behavioural tests were done to assess locomotor performance in malathion-treated mice. To evaluate the histopathological consequences in the mice brains, dopaminergic neurons in the substantia nigra were assessed by antibodies to tyrosine hydroxylase. Additionally, microglia and neuroinflammation were identified using Iba-1 immunostaining. We also investigated the role of dexamethasone (Dx) and acetyl salicylic acid (ASA) in ameliorating malathion neurotoxic effects. Results revealed that some neurobehavioural deficits were detected in malathion-treated mice. They were associated with decreased corpus striatum fiber density, increased dopaminergic neurodegeneration in substantia nigra and increased number of activated microglia. Treatment with ASA and Dx imparted significant protection against malathion neurotoxicity as evidenced by improved neurobehavioural performance and diminished neurodegeneration in the nigrostriatal system. Conclusion: Our findings advocate the use of antiinflammatory drugs as possible neuroprotective therapy against organophosphates-induced neurodegeneration. It is recommended to perform longer studies to examine the complete reversibility of OPs-induced neurotoxicity in response to different anti-inflammatory agents (the exact doses and duration) and to extrapolate the findings to humans chronically exposed to malathion.
Introduction
Pesticides are utilized for preventing, destroying, repelling or mitigating pests. They are also used in industry, therapeutics and warfare related products [1]. Organophosphate pesticides (OPs) are extensively used worldwide. Their high-intensity occupational application constitutes a global health problem due to the resultant environmental pollution and deleterious chronic health effects especially in developing countries [2,3].
It is worthy-mentioning that prolonged low level OPs exposure is not only limited to occupational settings, but also includes living with those occupationally exposed, living near OPs manufacturing and application sites or ingesting contaminated food [4].
The toxic effects of OPs include different phases i.e. acute toxicity, which involves acute cholinergic crisis due to accumulation of acetyl choline as a result of inhibition of cholinesterase (ChE) enzyme [5]. On the other hand, many reports stated that OPs neurotoxicity (especially in case of chronic exposure) may occur in the absence of ChE inhibition [6][7][8]. The exact clinical phenotyping of OPs-induced chronic neurotoxicity has been an area of debate with different suggested mechanisms [9,10].
Parkinson's disease is the most common neurodegenerative disorder that is accompanied by motor disability. The hall mark pathological finding in this disease is progressive degeneration of dopaminergic system, mainly the substantia nigra and corpus striatum [13]. The aetiology of sporadic PD (the most common form) is still not fully determined [14]. A current theory assumed that there is a gene-environment interaction, where genetic changes increase the vulnerability of dopaminergic neurons to risk of environmental toxic agents [15].
The mechanism of OPs-induced neurodegeneration is still unclear and seems to be different from AChE inhibition involved in acute toxicity. One suggested mechanism for OPs inducedneurotoxicity is neuroinflammation [9,10]. In the present study, we aimed to verify the degenerative effects of long term exposure to malathion on dopaminergic system in mice and to identify the possible contributing mechanism.
Material and Methods
This study was approved by Medical Research Ethics Committee, Faculty of Medicine, Mansoura University (code no: MD/106). All chemicals were purchased from Sigma -Aldrich ™ (Saint Louis, MO, USA) unless otherwise declared.
Animals and Experimental Design
This work was conducted in Medical Experimental Research Centre (MERC), Faculty of Medicine, Mansoura University, Egypt. First, a pilot study was performed to figure out the highest sublethal dose of malathion that produced no toxic clinical cholinergic signs, no morbidities as well as no mortalities. Three doses for malathion were tried (50, 100 and 200 mg/kg given to the mice by gavage).
To conduct the experiment, forty-eight BALB/c adult male mice (Albino inbred strains), and four month-old, 25-30 grams weight, were obtained from MERC. They were housed in clean cages under standard laboratory conditions including suitable temperature (22 ± 2°C), good lighting and aeration. They were fed a standard laboratory diet and tap water.
Mice were randomly divided into four groups (12 mice each) as follows: Control vehicle group (G1) that received distilled water (which is used to dissolve the treatments employed in the current study). Group 2 received malathion in a dose of 200 mg/kg dissolved in distilled water (1/2 ml). Malathion was administered orally by gastric gavage every day for 8 weeks. The other two groups received two different anti-inflammatory drugs (purchased from Amriya Company for pharmaceutical industries) in conjunction with malathion for 8 weeks as follows: Group 3 (G3): "malathion+dexamethasone" M+DX-treated group: mice were given malathion plus subcutaneous dexamethasone injection "3 mg/kg/day" [16] and G4: "malathion+acetyl-salicylic acid" M+ASA-treated group: mice were given malathion plus intraperitoneal injection of ASA "100 mg/kg/day" [17]. Weights of the animals were recorded once a week throughout the experiment.
Assessment of the Neurotoxic Effects of Malathion on Dopaminergic System
Locomotor performance tests Evaluation of the locomotor activity in mice was done at the end of the 8th week by using ANY-box ® (Stoelting Company, USA) which is a multi-configuration behaviour apparatus ( Figure 1). Two different behavioural tests were performed in a room that was completely isolated from external noises: Parallel rod floor test: Parallel rod floor test apparatus consists of a clear acrylic plastic box 20 cm × 20 cm × 30 cm (width, length, height) and a series of parallel stainless-steel rods placed on stainless steel base plate that acted as a floor for the chamber. The locomotor activity was assessed using two parameters: Foot slips (numbers of errors) which are measured by a touch sensor underneath the parallel rod floor in addition to efficient path (horizontal distance travelled by the mouse in cm).
Open-field test:
The apparatus is constructed of a clear acrylic plastic box 40 cm × 40 cm × 35 cm (width, length, height) fits to ANY-box base. Two perpendicular lines were drawn on the floor with a marker and were visible through the clear wall. These lines divided the floor into four equal quadrants: North East (NE), North West (NW), South East (SE) and South West (SW). Each animal was placed individually at the center of the apparatus, allowed to explore it freely and observed for five minutes. Each mouse trial was recorded for latter analysis, using a camera positioned above the apparatus. Locomotor activity was assessed for each mouse using numbers of mid zone cross, immobility, the number of entries into these quadrants and the duration that each animal spent in each quadrant. saline (PBS), followed by 150 mL of a cold fixative consisting of 4% paraformaldehyde, 0.35% glutaraldehyde, and 0.2% picric acid in 100 mM phosphate buffer (PB), under deep anesthesia with pentobarbital (100 mg/kg, i.p.). After perfusion, the brain was quickly removed and post-fixed for 2 days with paraformaldehyde in 100 mM PB and then transferred to 15% sucrose solution in 100 mM PB containing 0.1% sodium azide at 4°C. The brain pieces were processed into paraffin blocks, and then cut by microtome (20 µm). Antigen retrieval was done by emersion of the slides in ethylene diamine tetra-acetic acid (EDTA) solution for 20 minutes in a water bath at 90°C. Slides were then incubated with primary mouse monoclonal antityrosine hydroxylase (TH) antibody (diluted 1:1000) or ionized calcium binding adaptor molecule 1 (anti-iba-1 antibody: diluted 1:2000) over the night at 4°C in a refrigerator. After several washes by PBS, this was followed by biotinylated secondary antibody for 10 min then the avidin-biotin-peroxidase complex (ABC) 10 min at room temperature. All the sections were washed several times with PBS between each incubation, and labeling was then revealed by Diaminobenzidine (DAB) which was used as a chromogen. Slides were counterstained with Meyer's hematoxylin, dehydrated and cover slipped. Brain sections were examined with standard Olympus® light microscope (model-CX31RTSF).
Image analysis
Analysis was performed by a blinded investigator. The following areas were examined: Striatal TH-fiber density measurement: Pictures were captured by a digital camera (Olympus ® model E-420) and analyzed by using image J software version ij146-jdk6 for windows 7. Mean optical density of TH-positive dopaminergic fibers in the corpus striatum was assessed. To evaluate the entire striatal complex, the images were taken at six rostralcaudal levels corresponding to antero-posterior (AP): +1.60, +1.20, +0.20, -0.30, -0.90 and -1.40 mm from bregma. The striatum in each section was delineated and measured using the image J software. Non-specific background was correlated by subtraction of the non-specific binding as measured from the corpus callosum and for the TH-positive staining completely denervated areas of the striatum. The data represented the average of the six levels [18].
Dopaminergic neurons in Substantia Nigra pars compacta (SNpc):
TH-positive cells in the SNpc of both hemispheres were counted, in every fourth section, throughout the entire nucleus. The anatomical levels considered, in the anteroposterior (AP) extension, fell within -5.20 and -5.80 mm, with respect to bregma. Results were expressed as the percentage of cell loss compared to control. Neurodegeneration was ranked from one to four indicating loss of up to 25%, 50%, 75%, 100% in cell count respectively [19].
Microglia in nigrostriatal pathway: Microglial count in SNpc and corpus stiatum was evaluated using anti-Iba1 staining. Cells positive for Iba1 antibodies were counted in the same pattern as TH +ve neurons in both areas.
Statistical Analysis
The statistical analysis of data was performed using the computerized statistical package for the social sciences (SPSS) version 22.0 released 2013 and excel program for figures. Quantitative data was described as mean ± standard error of mean (SEM). For quantitative data; student t-test was used to compare between two groups. One way analysis of variance (ANOVA) test was used to compare more than two groups followed by Tukey's post hoc test. P is significant if<0.05 at confidence interval 95%.
Results and Discussion
Several researches reported a possible correlation between pesticides exposure and development of neurodegenerative disorders [3,20]. Since OPs are the most widely used pesticides, the previously mentioned studies focused on a link between OPs and PD which is the most common neurodegenerative disease. More interesting, some types of organophosphates have been proved to induce degeneration in the dopaminergic system [10,21].
In the present work, we investigate the neurotoxic effects of chronic malathion administration (an organophosphate compound that is widely used in Egypt) in BALB/c mice behaviourally and pathologically. Open field and parallel rod floor tests were used to assess exploratory behavior, anxiety, locomotor activity and motor incoordination [22].
We found that the change in the weights of the tested animals was insignificant. Some behavioural parameters were significantly affected in the Malathion group in comparison to the control mice. For instance, in parallel rod test, the efficient path was decreased (26.00 ± 2.45) and the number of foot slips was significantly increased in the malathion treated group (3.6 ± 0.24) in comparison with the control group (35.0 ± 1.67 and 0.9 ± 0.28 respectively). With respect to the open field test, the malathion treated mice had more North East entry frequency (2.6 ± 0.40) and they spent less duration in the South East area (27.6 ± 3.72) when compared to the control group (1.3 ± 0.30 and 45.6 ± 6.38 respectively).
It was reported that rats treated with malathion (50 and 100 mg/kg, for 3 days) presented anxiogenic behavior in the openfield test [23]. Additionally, Gould et al. [24] stated that reduced movement around the arena, and reluctance to enter or move from one place to another or spend time in the open central area indicated anxiety-related behaviour in the tested mice as observed in the current study.
It was necessary to verify the pathological impact of malathion on the dopaminergic system of the brains in the studied mice. Tyrosine hydroxylase (TH), an enzyme involved in the synthesis of dopaminergic neurons, was immunehistochemically labelled (TH+) to visualize the dopaminergic neurons and their neuronal processes. Furthermore, ionized calcium binding adaptor molecule 1 (Iba-1), a protein expressed by activated microglia was immunohistochemically labelled to detect microglial cell activation. The observed locomotor deficits were proved to be associated with evident degeneration in the dopaminergic system compared to the control group. Image analysis of corpus striatum showed significant degeneration in G2, treated with Malathion, compared to the control group as illustrated in Figures 2 and 3. The quantification of neurons in substantia nigra revealed parallel loss of dopaminergic neurons. Moreover, quantification of cells positive for Iba1 (microglia) showed accompanying increase in microglial cell count in parallel with microglial activation as evidenced by arborization of cells when examined under high power magnification (4 x) and this also paralleled the resultant neurodegeneration ( Table 1 and Figures 2 and 3). The results demonstrated in the present research are in accordance with previous findings showing dopaminergic degeneration due to chronic treatment with other types of organophosphates [25][26][27].
In order to verify the role of OPs induced-neuroinflammation in the current work, we assessed the therapeutic effect of two anti-inflammatory drugs (dexamethasone and ASA) behaviourally and pathologically. Our work revealed better performance in some parameters of the behavioural tests as well as significant improvement of the histopathological findings in G3 & G4 in comparison with the malathion exposed group despite they did not reach the same values of the control mice. Concurrent treatment with dexamethasone or salicylates reduced the number of activated microglia in striata of the treated mice compared to malathion alone treatment. In addition, the brain sections stained against TH showed significant decrease in the number of dopaminergic neurons in the substantia nigra. This cellular loss was parallel with decreased dopaminergic fibres density in corpus striatum. The decrease in microglia, hence neuroinflammation, was also accompanied with significant protection to dopaminergic system as evidenced by less degree of neurodegeneration and cell loss.
Thus, anti-inflammatory drugs administration in conjunction with malathion improved the behavioural dysfunction in malathiontreated mice and was associated with decreased levels of anxiety. Interestingly, anxiety has been reported to be a predominant clinical symptom of neurodegenerative diseases [28]. Therefore, dexamethasone and ASA may have a protective effect in the treatment of neurodegenerative disorderassociated anxiety. Besides, idiopathic PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta leading to dopamine depletion [29] which is also improved after administration of these drugs as demonstrated in the current research.
One important area that correlates OPs and PD is the mechanism of action. Some studies revealed that one of the mechanisms of dopaminergic degeneration is through neuroinflammatory mediators [30,31]. The proinflammatory cytokines and cells play crucial role in the damage of vulnerable dopaminergic cells. On the other hand, several reports showed that OPs induce chronic neurotoxicity through induction of neuroinflammation and could also induce dopaminergic damage through their proinflammatory effects [21,32,33].
In the present work, malathion treated mice showed significant elevation of microglial count and activation in corpus striatum. Worthy mentioning, microglia is the resident immune cells (macrophages) of the brain which can be triggered and activated in response to proinflammatory trigger or neuronal death. Then, several reactive oxygen species and proinflammatory factors (e.g., tumor necrosis factor α, interleukin-1β) are produced and in turn, contributing to neurotoxicity and degeneration [34,35]. In this context, OPs including malathion may initiate an inflammatory response leading to the activation of microglia as noticed in the current work.
These findings denote proinflammatory mechanism of malathion induced neurodegeneration which support previous reports on OPs mechanisms of neurotoxicity and aetiological pathways for dopaminergic system damage [36,37].
One limitation of our study is the lack of assessment of cholinesterase levels. However, it is known that the decreased AChE activity does not correlate with the observed symptoms or reported deficits and manifestations of exposure persist long after ChE levels return to normal [8,38].
In conclusion, the present findings denote the ability of OPs to damage dopaminergic system. Moreover, microglia activation indicates that these effects may be related to malathion induced dopaminergic degeneration and could be induced by neuroinflammation.
Targeting inflammation offered neuroprotection in our work, this may advocate the use of antiinflammatory drugs as therapeutic strategy. Further longer studies are warranted to examine the complete reversibility of OPs neurotoxicity in response to different anti-inflammatory agents (the exact doses and duration) and to extrapolate these findings to humans chronically exposed to malathion. | 2019-04-02T13:11:29.659Z | 2017-01-01T00:00:00.000 | {
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105571481 | pes2o/s2orc | v3-fos-license | Optimal formulation of a product containing black wheat granules
ABSTRACT This study describes the development process of a healthy nutritional product containing black wheat granules (BWGP), which was based on Xinjiang characteristics of miscellaneous grains and nuts. BWGP was designed and developed as a local characteristic of nutritious food, which is produced from wheat, black sesame seeds, flour, corn flour, chickpeas, walnuts, peanuts, melon seeds, red dates, and brown sugar. The product was then underwent sterilization process followed by the inflated or vacuum quantitative packing as an instant ready-to-eat food. The best formula (%) was determined as follows: black wheat 30, corn flour 20, flour 15, chickpeas 15, black beans 2, peanuts 3, melon seeds 3, walnut 3, black sesame seeds 4, red date powder 15, sugar powder 15, maltodextrin 15, and sunflower oil 20. BWGP has several advantages, which include: a regional characteristic, simple production methods, and rich flavors. Overall, our study focused on the nutritional, flavor, and compositional effects and how a food product can be made healthier, more sustainable or more acceptable to the consumer.
Introduction
In the 21st century, consumers are no longer simply satisfied with the daily food [1] , but start to focus on eating well and healthy. [2] Therefore, consumers have begun to bring food together in a comprehensive, diverse, and borderless manner. [3] A "food and nutrition, eating healthy" food revolution is quietly beginning all over the world. [4] There is a growing understanding of the relationship between food ingredients, diet, and health among consumers who are aware of the effect of specific food ingredients on health and physiological functions. [5,6] Currently, there are many kinds of powdered foods that are readily available on the market, [7] such as black sesame paste, walnut powder, and lily powder. However, most of these instant granules use only a small amount of the main ingredients, and many of these products are similar to each other. In the Xinjiang Uygur Autonomous Region, China, there are some characteristic food ingredients that can be used to develop a healthy nutritional granulated product. [8] One of the food ingredients used in the design of Xinjiang characteristic granulated product is black wheat (triticale), which is rich in protein and antioxidant. Black wheat also contains highquality proteins and essential amino acids, particularly the content linoleic and linolenic acids are 30%-50% higher than normal wheat. Its consumption is associated with several health benefits such as protecting endothelial cells, preventing heart and cardiovascular diseases (CVD) and as anticancer agents. [9,10] In addition, it also contains anthocyanins and procyanidins. These bioactive compounds are associated with a reduced risk of the pathogenesis of chronic diseases such as diabetes, obesity, cancer, and cardiovascular disease. [11,12] Other ingredients such as sunflower oil are in the design of Xinjiang characteristic granulated product. The oil content of sunflower oil is generally between 30%-70% [13] , in which linoleic acid content is usually around 65%-73%. [14] Since the linoleic acid content is high, it is associated with some health benefits such as a reduction of blood pressure and serum cholesterol. [15] Moreover, sunflower oil is also rich in vitamin A, D, and E. [16,17] Therefore, regular consumption of sunflower oil is associated with a reduced risk of atherosclerosis, hypertension, and coronary heart disease.
Chickpeas (Cicer arietinum L.) are also used in the design of Xinjiang characteristic granulated products. Chickpeas are one of the traditional sources of protein intake in humans. They are usually fermented or fried, especially in India, Spain, and some Mediterranean countries. Chickpeas have become the world's third most important food legumes after dry beans and dried peas because the global consumption of chickpeas has increased steadily in recent years. [18] Chickpeas are rich in potassium and phosphorus. [19] For those who are lactose-intolerance, chickpeas can account for a large proportion of calcium required. Chickpeas are a good source of B vitamins, vitamin C, A, E, and K. Chickpea protein content ranges from 18% to 28%. [20,21] Its protein digestibility, biological value, net protein utilization, and protein efficiency are higher than soybeans. [22,23] Even with different methods of processing chickpea, this does not affect the composition of its amino acids. [24] This can greatly improve the diet of malnourished people. The protein digestibility of chickpeas ranges from 48% to 89.01% [25] , higher than that of soybeans and peas. [26] Chickpeas can also lower cholesterol level because of the composition of its fatty acids. Chickpeas have 35% oleic acid and 63% linoleic acid. [27,28] Other food ingredients such as black sesame and jujube (Chinese date) are also used in the design of Xinjiang characteristic granulated products. Black sesame is rich in vitamin E content [29,30] and its consumption has been associated with health benefits such as protective against CVD and hypertension. [31,32] Jujube is high in sugar content. [33] It is rich in various proteins, amino acids, carotenoids, vitamin C, vitamin B 2 , calcium, iron, and phosphorus. [34] Therefore, the aim of the study was to develop a healthy nutritional product containing black wheat granules (BWGP), which was based on Xinjiang characteristics of miscellaneous grains and nuts.
Materials and reagents
Black wheat, black beans, black sesame, chickpeas, corn flour, flour, peanuts, melon seeds, walnuts, red dates, and brown sugar powder were bought from Urumqi Home Supermarket Co., Ltd. (China). Additives including maltodextrin, calcium carbonate, calcium gluconate, vitamin C, TBHQ, BHT, and D-VC sodium were provided by the manufacturers and complied with the Chinese National Standards.
Operation points
Black wheat, flour, and corn flour were roasted in a sealed pot at 80-100°C for 10-15 min in order to remove excess moisture from cereals. Then, the mixture was heated up to 150-160°C for 15 min in order to produce starch paste and flavors.
Roasting of black wheat, flour, and corn flour
The aroma of the mixture gradually appeared with increasing roasting time. Different roasting times were applied on black wheat, flour, and corn flour. When the roasting time was increased, the color of black wheat, flour, and cornflour changed gradually from white to yellow and finally to brownish. The wheat flavor was also increased with time.
Baking of black wheat, flour, and corn flour
Using 80-100 mesh sieve of fine powder as raw materials, the mixture was baked using an oven.
Pre-treatment of black beans
Fresh black beans were smashed using a universal crusher with 80-120 mesh sieve. Before adding to the mixture, black bean powder was stir-fried to increase its aroma.
Black sesame treatment process
High-quality black sesame seeds were soaked in the water to remove the soil and sediment. Then, black sesame seeds were blast dried to remove excess moisture on the surface of black sesame seeds. Black sesame seeds were baked on an ACA oven. After black sesame seeds were stir-fried using a flat pan at 160-180°C for 10-15 min, they were quickly cooled down for the esterification reaction to take place. Figure 1. Shows the production process flow of BWGP from raw materials screening, processing, mixing, semi-finished products, testing, packaging, finished product sales, and other links.
Pretreatment of chickpeas
After fresh chickpeas were roasted using an oven, the chickpeas were smashed by a universal crusher with 80-120 mesh sieve.
Pretreatment of red dates
Red dates were graded according to their quality red dates before they were washed to remove the impurities. After removing pits from red dates, they were cut into small pieces for roasting at 60°C for 12 h to reduce the water content to <4%. Then, they were smashed using a universal crusher with 80 mesh sieve.
Results and discussion
Effects of baking condition on sensory evaluation of wheat flour In Fig. 2, when baking at 140°C for 40 min and at 160°C for 30 min, the color of wheat flour gradually changed from white to dark yellow and then brown; and the aroma was also richer; the taste also had also changed from raw flour flavor into a slightly sweet cooked flour flavor, and there was a little bit of clumping. In addition, the color, taste, and aroma were good in all aspects. [35] Since a longer baking time can have a certain impact on the equipment and increase the cost of production, it is better to choose the baking temperature of 160°C and the baking time of 30 min.
Effects of baking conditions on sensory evaluation of flour
In Fig. 3, when baking at 160°C for 40 min and 180°C for 5-10 min, the color of flour gradually changed from white to yellowish, and the aroma was also richer. The initial raw flour flavor gradually turned into a slightly sweet pasta flavor. Also, flour also caused agglomeration because of an increase in water loss. Although the color, taste, and aroma of flour were better in all aspects at 160°C for 40 min, a shorter baking time is preferable when taking into consideration both the food production and processing processes. Therefore, it is advisable to choose the baking temperature of 180°C and baking time of 15-20 min.
Effects of baking conditions on sensory evaluation of corn flour
In Fig. 4, when baking 160°C for 30-40 min and 180°C for 5-10 min, the color of corn flour gradually changed from light yellow to yellow-brown; the aroma of corn was obvious; the taste of cornflour gradually turned into a slightly sweet taste of cooked corn. Corn flour was different than flour, even when there was a large water loss, and basically, no agglomeration was observed and sensually as soft as fine sand. Although corn flour was better in color, taste, and aroma at 160°C for 30-40 min, the baking time was too long and it was not efficient for the cost of food production and processing processes. Therefore, it is advisable to choose the baking temperature of 180°C and baking time of 5-10 min.
Effects of baking conditions on sensory evaluation of black beans
When an excessive amount of black bean powder was added, this would give a more intense smell of black beans and will dilute the aroma of sesame and red dates. In Fig. 5, when baking at 140°C for 25-30 min, it gave the unique aroma of black beans. However, when the temperature was too high or baking time was too long time, black beans gave a sense of burnt and this would greatly affect the taste of the product, Therefore, it is advisable to choose the baking temperature of 140°C and baking time of 25-30 min.
Selection of roasting, baking conditions and content of black sesame seeds
Black sesame seeds were baked before crushing in order to remove the bitter taste of black sesame outer layering and increase the aroma of black sesame seeds. Stir-frying black sesame seeds gave an outstanding aroma fragrance and if followed by crushing (more than 40-60 mesh), the aroma would be particularly prominent. However, it is not recommended to grind them too fine because this would affect the overall taste. In addition, once the black sesame seeds are cooked, they should be used as soon as possible. Their storage time should not be too long because the unique aroma of the black sesame seeds would gradually dissipate.
Effect of baking conditions on sensory evaluation of black sesame seeds (Fig. 6). In Fig. 6, a too high baking temperature would make the black sesame seeds to produce a char smell. At the same time, the color of black sesame also turned from black to gray, and this would greatly affect the taste and color of products. Therefore, the most suitable condition was determined to be 160°C for 20 min.
Effects of added black sesame on the sensory quality of BWGP
In Table 1, the color, aroma, and taste of the BWGP increased with the addition of black sesame. However, when the dosage was more than 5%, the aroma of black sesame concealed the aroma and taste Figure 6 is the same as t Figure 2. of other materials, which was similar to that of black sesame paste. When the dosage was less than 3%, the black sesame aroma was lacking and this caused the fragrance not sufficient. Therefore, after considering the distinction between the ubiquitous black sesame pastes available in the market, the product's fragrance, the characteristics of Xinjiang foods, product sale costs and practical application of operations, the addition of 3%-5% black sesame seeds was considered to be appropriate. [36] Effects of roasted chickpeas on the sensory quality of BWGP Chickpeas were baked by an ACA oven, but the effect was not better than frying. This is because frying can heat the chickpeas more evenly and quickly while avoiding chickpeas get burnt. The temperature of frying was 140-160°C for 20 min. Cooked chickpeas basically had no bean flavor. Therefore, an additional amount of chickpeas could be added as appropriate. However, considering that the poor water retention of chickpeas and the homogeneity of products, the amount of chickpeas added was determined to be 20%.
Effects of red dates on the sensory quality of BWGP
Adding a certain amount of red date powder to BWGP not only improved the sweetness of the product in synergy with brown sugar; but also made the product rich in aroma due to the special thick aroma of red dates. In Table 2, the sweetness, aroma, and taste of BWGP increased with the addition of the amount of red dates. With increasing amount of red dates added, the flavor of red dates was stronger, while the sunflower, sesame, peanut, and other fragrance were diluted. The flavor of red dates was too strong, vaguely accompanied by bitterness. Therefore, it was more appropriate to add 15% of red dates. Table 3, when the amount of brown sugar was 5%, the sweetness was obviously insufficient. When it was added to 20%, there was a sweet feeling (except for those who had sweet tooth). When the amount of sugar was about 15%, moderate sweetness, in line with popular preferences, the comprehensive evaluation of the selection of brown sugar to add about 15% was appropriate.
Sensory assessment
Before products are released into the market, a sensory assessment is needed to be performed. However, the sensory assessment is easily to be affected by the external environment and human subjective factors which will result in a lack of certain objectivity and biased results. [37] Therefore, the experiments are based on fuzzy mathematics theory in order to optimize the sensory test, quantify the ambiguity, the external environment, and subjective factors. [38,39] This is a more scientific and accurate method to do the sensory evaluation. [38,39] It also provides a theoretical basis for the industrial application using rational formulas. [38,39] Determination of domain assessment The assessment domain related to the scopes of the study was determined in the form of a collection (a factor in which a branch of a domain is interrelated and has some relevance). In the sensory evaluation, the assessment domain is usually used as a general set of indicators that best reflect the quality of the food. Generally, this is referred as: U, U = {u 1 , u 2 , u 3 , . . ., u n }; where u i represents the corresponding evaluation index of the i-th item, denoted as "i = 1,2,3,. . . n". The organizational structure, color and luster, aroma and taste, impulse stability and taste were the important sensory indicators of BWGP, which provide a more comprehensive reflection of the BWGP sensory quality. Therefore, these five indicators were selected as the evaluation objects and sensory evaluation was carried out by the method shown in Table 1. For example, u 1 was set to represent the organizational structure, u 2 was the color and luster; u 3 was the aroma and taste, u 4 was the impulse stability, and u 5 indicates the taste. The final output for BWGP was calculated as U = {u 1 , u 2 , u 3 , u 4 , u 5 }.
For the sensory evaluation, a total of 10 participants were selected based on criteria including tastesensitive in the research direction of agricultural product processing. According to the sensory evaluation criteria, the five indicators of BWGP were evaluated and the scores were recorded by participants.
Determination of the commentary domain scores
The comment domain refers to the set of feedback information about the evaluation index of a certain kind of food. Comments can be given in the form of text and used to represent a certain value or level. Usually, the commentary of the sensory test is denoted as: V, V = {v 1 , v 2 , v 3 , . . ., v n }; where v i represents the evaluation level or score of the corresponding item i, Remember: i = 1, 2, 3, . . ., m. According to the quality rating criteria shown in Table 1, the comment on the Black Wheat nutrition granules was calculated as: V = {v 1 , v 2 , v 3 , v 4 }, Namely: V = {excellent, preferably, general, unqualified}. The sense of taste is relatively adequate, but the former taste is lacking Taste obvious, before and after the taste is obvious, especially after the taste The taste is obvious, the former taste is full, but not a favorite after feeling
Determination of the weight vector
Due to the different degree of influence of the indicators on quality, a weight vector can be determined according to the weight value of each index. The weighting factors are: A = (a 1 , a 2 , a 3 , . . ., a n ), 0≤ a i ≤ 1, ∑a i = 1, where A is a fuzzy subset of U; A and U are corresponding to each other, and A is a subset of U. The weight vector related to triticale BWGP is obtained according to the weight values of the indexes in Table 4.
Establishment of the quality factor weight set
User survey method was adopted in the study. [40] A ratio of 1:5 or 1:6 above 80°C hot water was added to 35-45 g BWGP samples, which were given to the 10 participants who did the sensory evaluation based on the five indicators. [41] The weight of each quality factor, K was determined for the five indicators. The results were as follows: organizational structure (0.215), color and luster (Table 5).
Establishment and results of fuzzy judgment matrix Table 5 shows the results of 10 participants according to the BWGP sensory evaluation criteria. The 5 indicators of the sample were determined by the 8 groups of samples (i.e., 8 sets of samples were different in the formulation adjustment) and the distribution of votes for each factor in each level was determined (Table 6). Fuzzy matrix 1-8 samples are as follows: Fuzzy transformation and a comprehensive score The weight K was multiplied by the fuzzy relation matrix A j , namely: R j = K× A j . For example, corresponding to j sample, the evaluation result was R j which reflected the excellent, preferably, general or unqualified approval rate of sensory evaluation for sample j. Taking the first group of samples as an example, the results of comprehensive evaluation of the samples were as follows: Table 6. BWGPs for sensory evaluation of the distribution of votes (Fuzzy Judgment Matrix).
Organizational structure Color and lustre Aroma and taste Impulse stability Taste The results showed that, under the condition that the content ratio was adjusted in different formulations, all participants reported that the treatment of BWGP under this condition was well. This indicated that the sample no. 8 had a favorable rating of 34.1%; 23.95% considered it as better; 41.95% considered it as normal and none considered this as failed. Similarly, the results of the comprehensive score of samples no. 2-8 were as follows: Combined with the sensory evaluation of fuzzy mathematics, 8 samples were evaluated and evaluated by 10 participants in order to establish a comprehensive score. By comprehensive score: The unqualified rates of 1st, 2nd, 5th, and 7th samples were between 10% and 22%, suggesting a relatively high product failure. While the 3rd and 6th samples had a positive rate of 8.35% and 16.35%, respectively. The sample no. 8 had a qualified rate of 100%, and the unqualified rate was 0. Meanwhile, the sample no. 4 also came after group 8th with higher scores and a relatively lower 5.9% rejection rate.
Using the formula, samples with the best ratio (i.e., the best quality of BWGP) were considered as the highest quality of the fuzzy sensory scores. Therefore, sample no. 8 had the most favorable ratio, reaching 34.1%, and the sum of ratios in favor reached 58.05%. Also, none think that the quality of the sample No. 8 was failed. Therefore, according to the BWGP fuzzy mathematical sensory comprehensive score, the sample no. 8 was corresponded to the processing conditions that indicated the best formula match.
Conclusion
From the selection of the various ingredients in the BWGP to the two methods of baking and frying the raw materials, the fuzzy evaluation of BWGP was carried out by the fuzzy mathematics sensory evaluation method. The best formula weight was set up, and the best formula weight set was K = (organizational structure 0.215, color and luster 0.195, aroma and taste 0.190, impulse stability 0.20 and taste 0.20). Therefore, sample no.8 was the best BWGP formula. The best formula (%) was determined as follows: black wheat 30, corn flour 20, flour 15, chickpeas 15, black beans 2, peanuts 3, melon seeds 3, walnut 3, black sesame seeds 4, red date powder 15, sugar powder 15, maltodextrin 15, and sunflower oil 20. Therefore, the development of BWGP will promote the use of some characteristic food ingredients in Xinjiang Uygur Autonomous Region, China because the expanded utilization of BWGP will increase its production. This will result in the social development of the region by creating job opportunities and opening new markets. Since the success of this study showed that BWGP is nutritious and easy to be prepared, future study should determine if BWGP can be used to provide food security, nutrition, and sustainability in populations. | 2019-04-10T13:11:52.458Z | 2018-01-01T00:00:00.000 | {
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246437768 | pes2o/s2orc | v3-fos-license | Description and molecular characterisation of Pelecitus copsychi Uni, Mat Udin & Martin n. sp. (Nematoda: Onchocercidae) from the white-rumped shama Copsychus malabaricus (Scopoli) (Passeriformes: Muscicapidae) of Pahang, Malaysia
Species of the genus Pelecitus Railliet & Henry, 1910 the most widely distributed avian filariae in Africa and South America. Zoonotic cases in humans were reported in South America. While investigating the filarial fauna of wild animals in Malaysia, we discovered an undescribed filaria from the swollen footpad of the left leg of Copsychus malabaricus (Scopoli) in Pahang, Peninsular Malaysia. Adults of both sexes have a corkscrew-shaped body. Based on comparison of their morphological characteristics (i.e. pre-oesophageal cuticular ring distinct, oesophagus divided, vulva protuberant and situated at the level of anterior half of oesophagus, spicules strongly sclerotized and left spicule with broad blade) with other Pelecitus species, they are here described as Pelecitus copsychi Uni, Mat Udin & Martin n. sp. Multi-locus sequence analyses based on seven genes (12S rDNA, cox1, 18S rDNA, 28S rDNA, MyoHC, rbp1 and hsp70) were performed to determine the phylogenetic position of the new species. The calculated p-distance between the cox1 gene sequences for P. copsychi n. sp. and Pelecitus fulicaeatrae (Diesing, 1861) was 14.1%. Intraspecific genetic variation between two individuals of the new species was 0.4%. In both the Bayesian inference and maximum-likelihood trees, P. copsychi n. sp. was positioned in the second clade of ONC5, containing three genera of the subfamily Dirofilariinae (Foleyella Seurat, 1917, Pelecitus and Loa Stiles, 1905). Immunostaining and molecular analyses remained negative for the presence of Wolbachia endosymbionts. Our findings corroborate the division of the subfamily Dirofilariinae into ONC3 with Dirofilaria Railliet & Henry, 1911 and ONC5 with Pelecitus.
The pathology caused by Pelecitus spp. in their avian hosts includes swollen joints, lameness and chronic inflammatory tenosynovitis (Greve et al., 1982;Allen et al., 1985;Bartlett, 2008). Adult worms are generally localized in the tendons of the legs and feet, sometimes in the muscles of the legs or in the articulations of the tibia or knee joint (Bartlett & Greiner, 1986; Supplementary Table S1). Recently, Muñoz-García et al. (2018) reported a severe tenosynovitis caused by Pelecitus sp. found in swollen nodules on both legs of a crested caracara Caracara cheriway (von Jacquin) with signs of pain in Mexico. Rare cases of zoonotic infections caused by Pelicitus sp. have been described in humans (Bortero et al., 1984;Bain et al., 2011). Bain et al. (2011) reported Pelecitus sp. from the iris of a man in Brazil and suggested that specimens that had been recovered from the anterior chamber of the eye of a patient in Colombia and identified as Loaina sp. by Bortero et al. (1984) belonged to Pelecitus as well.
In Indomalaya, Pelecitus ceylonensis Dissanaike, 1967 was recovered from two naturally infected crows (Passeriformes) as well as from experimentally infected ash doves (Columbiformes) and chickens (Galliformes) in Sri Lanka (Dissanaike, 1967;Supplementary Table S1). In addition, Pelecitus galli Dissanaike & Fernando, 1974 was described from Gallus gallus spadiceus (Bonnaterre) (Galliformes) in Tasek Bera, Pahang, Malaysia (Dissanaike & Fernando, 1974). Although 36 species of filarial parasites from 22 genera have previously been recorded in vertebrates in Malaysia (Yen, 1983;Gibbons, 2010;Uni et al., 2017Uni et al., , 2020, P. galli is the only species of Pelecitus reported in Malaysia to date. During a scientific inventory of the Krau Wildlife Reserve, Pahang, Malaysia, organized by the Department of Wildlife and National Parks, Malaysia in 2013, we collected nematodes from the swollen footpad of the leg of a white-rumped shama Copsychus malabaricus (Scopoli) (Passeriformes) caught in the primary forest. Herein, we describe the specimens as Pelecitus copsychi Uni, Mat Udin & Martin n. sp. and provide their molecular characterisation, revealing a close association with P. fulicaeatrae from birds and Foleyella candezei (Fraipont, 1882) from squamates in Africa (Bartlett, 1986).
A recent study on the phylogenetic relationships within the family Onchocercidae included few species of avian filariae, namely Aproctella alessandroi Bain, Petit, Kozek & Chabaud, 1981, Cardiofilaria pavlovskyi Strom, 1937 and P. fulicaeatrae (see Lefoulon et al., 2015). In the present study, newly generated as well as previously published partial sequences of two mitochondrial (12S rDNA and cox1) and five nuclear genes (18S rDNA, 28S rDNA, the myosin heavy chain (MyoHC), RNA polymerase II large subunit (rbp1) and 70 kDa heat-shock proteins (hsp70)) were used to study the phylogenetic interrelationships within the Onchocercidae, including a wider range of avian filariae.
Collection of hosts and parasites
A scientific inventory, organized by the Institute of Biodiversity, Department of Wildlife and National Parks, Malaysia, in Sungai Teris (3 36 0 42.6 00 N, 102 06 0 55.9 00 E), Krau Wildlife Reserve, Pahang, Malaysia, was undertaken between 21 May 2013 and 27 May 2013. During this inventory, a white-rumped shama (ID no. A8) with a swollen footpad of the left leg was examined for parasites. The swollen footpad was dissected under a stereomicroscope and 16 filarial parasites (12 females and 4 males) removed for subsequent morphological and molecular analyses.
Morphological study
Adult worms (8 females and 4 males) were fixed in 70% ethanol for morphological examination. Specimens were cleared in lactophenol (R & M Chemicals, Essex, UK) and drawn under a compound microscope equipped with a camera lucida (Olympus U-DA, Olympus, Tokyo, Japan). Measurements are in micrometres unless otherwise indicated and are given as the range; body length was measured following the helical coils. Thick blood smears were made and stained with 3% Giemsa solution (pH 7.4). One female fixed in 70% ethanol was embedded in paraffin, and sections were stained with haematoxylin and eosin (HE).
Samples were sequenced at the Mus eum National d'Histoire Naturelle (MNHN), using a MiSeq Illumina. Libraries were prepared following the protocol of Meyer & Kircher (2010). The reads were paired-ends with a theoretical size of 250 bp. For each gene, reads were mapped against the P. fulicaeatrae sequence (Supplementary Table S2) using Bowtie2 v2.4.1 (Langmead & Salzberg, 2012) with the following parameters: seed length of 18 bp; "very sensitive local" mode; mixed or discordant pair-end reads not allowed. The mapped reads were excluded if their length was inferior to 75 bp, or if more than 1% of their length was soft-clipped or if their edit distance was superior to 5% of their length. The remaining reads were assembled against their sequence of reference using IGV v2.9.4 (Robinson et al., 2011).
Filarial cox1 gene analysis
A DNA barcoding approach based on the cox1 marker was used to identify the specimens of Pelecitus from C. malabaricus. The cox1 marker was chosen over the 12S rDNA marker for its greater robustness (fewer indels, less susceptible to dataset variation) (Ferri et al., 2009). The cox1 sequence divergence was estimated by the number of base differences per site between two sequences (p-distance) using MEGA version 11 (Tamura et al., 2021). Cox1 sequences generated during the present study and those from the literature were analysed. Pairwise comparisons between the selected 48 cox1 sequences were processed, with each sequence representing a species (Supplementary Table S3).
Phylogenetic analyses
Sequences generated during the present study and those from the literature (Supplementary Table S3) were aligned using SATe v2.2.7 (Liu et al., 2009). Fifty-one sequences for 50 species belonging to 27 genera were included in the analyses and their phylogenetic relationships inferred using Bayesian inference as implemented in MrBayes 3 (Ronquist & Huelsenbeck, 2003). A partitioned model was implemented to estimate evolution parameters separately for each gene. For the analyses, the weighted average of the best-fit evolution models in the GTRþG landscape was used for each gene. Two runs were performed using five million steps with four chains, with tree sampling every 1000 generations; the first 25% of the trees were discarded as "burn-in" and posterior probabilities were calculated from the remaining trees.
In addition, a phylogeny based on the maximum-likelihood (ML) was implemented. For each gene, the best-fitting substitution model was determined using the corrected version of the Akaike Information Criterion (AICc) in JModelTest v2.1.10 analyses (Guindon & Gascuel, 2003). The TPM1ufþIþG was the best fit for 12S rDNA; TPM1þIþG for 18S rDNA; TVMþIþG for 28S rDNA; GTRþIþG for cox1; TPM2ufþIþG for hsp70; TrNþG for MyoHC and TPM3ufþG for rbp1. To root the trees, two species were included as the outgroup: Filaria latala Chabaud & Mohammad, 1989 (Spirurida: Filariidae) and Protospirura muricola Gedoelst, 1916 (Spirurida: Spiruridae). The phylogenetic relationships of the Onchocercidae were inferred by the ML on the partitioned concatenated dataset. The program was executed by generating 10 random start trees with 1000 bootstraps using RaxML-NG (Kozlov et al., 2019).
Wolbachia detection
Sections of a female of P. copsychi n. sp. were stained with a rabbit polyclonal antiserum raised against the surface protein of Wolbachia from Brugia pahangi (Buckley & Edeson, 1956) (1:2000 dilution) as described by Kramer et al. (2003) and Ferri et al. (2011).
Site in host: Adult worms occurred free in the swollen footpad of the left leg.
Etymology: The specific epithet is derived from the genus name of the type-host.
Description
General.
[ Table 1; Figs. 1 and 2.] Body corkscrew-shaped in both sexes, in form of dextral helix with 3-4 rotations (Fig. 1A). Body width uniform over most of length but tapering gradually at both ends. Cephalic papillae arranged in four submedian pairs, not markedly protuberant. Amphids small, lateral, not salient. Pre-oesophageal cuticular ring present and distinct (Figs. 1B and 2B). Oesophagus clearly divided into anterior muscular and posterior glandular portions ( Fig Microfilaria. [Based on 10 specimens from anterior parts of uteri.] Loose sheath present, extending past anterior extremity of body. Extremities bluntly rounded ( Fig. 2E and F). Microfilariae not found in thick blood smears of infected host. Male.
Remarks
The present specimens were assigned to the genus Pelecitus (Onchocercidae: Dirofilariinae) as defined by Anderson & Bain (1976), Bartlett & Greiner (1986), Bartlett & Anderson (1987b) and Gibbons (2010), because of their corkscrew-shaped body, the presence of a pre-oesophageal cuticular ring and lateral alae that extend from the cervical region to the caudal extremity. Although not observed in males, a single post-deirid was found in females. Microfilariae are bluntly rounded at both extremities and possess a loose sheath. As is typical for the genus, the present specimens were associated with tendons and muscles near the leg joints of their host. To date, 17 species of Pelecitus have been recorded from birds (Bartlett & Greiner, 1986;Bartlett & Anderson, 1987a;Spratt, 2010;Supplementary Table S1).
We compared the morphological characteristics and morphometrics of the present specimens with three congeners reported in Indomalaya and Australia: P. ceylonensis in Sri Lanka, P. galli in Malaysia, and P. bartneri in Australia (Table 1). Pelecitus copsychi n. sp. is distinct from P. galli and P. bartneri in that its females are shorter, only reaching half the length of the latter two species. In contrast, both males and females of P. copsychi n. sp. possess a longer oesophagus when compared to the remaining three species. In addition, the oesophagus is clearly divided into a muscular and glandular portion in the new species, but not in P. ceylonensis or P. galli. While in the present specimens, the vulva is located at the level of the anterior half of the oesophagus ( Fig. 2A), it is located at the level of the posterior half in the remaining species (Table 1). Tail length of the present females is greater than that of P. ceylonensis, but shorter than that of P. galli and P. bartneri. Furthermore, the microfilariae of P. copsychi n. sp. are somewhat shorter than those of the remaining species.
The present species can be further differentiated from P. galli in that its males are shorter and more slender at midbody, but with a tail that is longer than that of P. galli. In P. copsychi n. sp., granular or hyaline inclusions were not seen in the caudal alae of males, but such inclusions are present in the caudal alae of P. bartneri (see Spratt, 2010).
Pelecitus fulicaeatrae has a wide host and geographical range. The species has been recorded in Fulica atra L. (Gruiformes) and various other hosts from England, Germany, Russia, Japan, France, Australia, Canada and Madagascar (Supplementary Table S1). However, P. copsychi n. sp. can be readily distinguished from this cosmopolitan species in that in P. fulicaeatrae, the pre-oesophageal ring is delicate, the oesophagus is not divided, and the vulva is situated at the level of the posterior half of the oesophagus, close to or posterior to the oesophago-intestinal junction (Vanderburgh et al., 1984;Bartlett & Greiner, 1986;Spratt, 2010).
The present species is further distinguished from the remaining 13 congeners parasitizing avian hosts by a combination of characters (Bartlett & Greiner, 1986;Bartlett & Anderson, 1987a). With the exception of Pelecitus vuylstekae Vuylsteke, 1957 and P. copsychi n. sp., in which the vulva in females is at the level of the anterior half of the oesophagus, the vulva is positioned at the level of the posterior half of the oesophagus in the remaining species. However, P. vuylstekae can be distinguished from the new species in that its females (19-25 vs 4.5-8.7 mm) and males (8-9 vs 4.5-4.6 mm) are longer, its spicules are not strongly sclerotized, the blade of the left spicule is slender, and inclusions are occasionally present in the caudal alae (Bartlett & Greiner, 1986). In the pre-oesophageal ring being delicate or absent, Pelecitus andersoni Bartlett & Greiner, 1986, Pelecitus chabaudi Bartlett & Greiner, 1986 (Bartlett & Greiner, 1986). In addition, the vulva is only slightly or not protuberant in P. andersoni, P. chabaudi and P. helicinus, while it is markedly protuberant in P. copsychi n. sp. Furthermore, caudal inclusions are present in P. andersoni, P. helecinus, P. polamaetus and P. tubercauda (see Bartlett & Greiner, 1986), but absent in the present specimens. Additional distinguishing features between these congeners and the new species are the oesophagus being undivided or only indistinctly divided in P. chabaudi, P. circularis and P. tubercauda and the weakly sclerotized spicules in P. polamaetus, with a narrow blade seen in the left spicule (Bartlett & Greiner, 1986), as opposed to a clearly divided oesophagus and well-sclerotized spicules with a broad left blade in P. copsychi n. sp. Pelecitus anhingae Vuylsteke, 1957 and Pelecitus tercostatus (Molin, 1860) are similar to the new species in that the pre-oesophageal ring is readily apparent and the oesophagus is divided (Bartlett & Greiner, 1986). However, in the former two species, the spicules are not well-sclerotized and the left spicule has a narrow blade (Bartlett & Greiner, 1986). A further congener in birds, Pelecitus armenica Chertkova, 1945 can be distinguished from the new species by females being longer (15.5 mm) and males having longer spicules (left spicule 150 μm and right spicule 110 vs 80-90 μm and 62-73 μm, respectively) (Bartlett & Greiner, 1986). Bartlett & Greiner (1986) considered Pelecitus barusi Coy Otero, 1982 morphologically very close to P. galli, but postponed a decision on their synonymy until more material of the former species could be examined. Pelecitus barusi differs from the new species in the females being larger (15-20 mm long) and the oesophagus being undivided or indistinctly divided (Bartlett & Greiner, 1986).
Little information is available on P. major and P. spiralis from Passeriformes in Nigeria. However, given their host spectrum (Ploceidae) and geographical distribution in Africa (Bartlett & Anderson, 1987a), it is unlikely that they should be conspecific with our material collected from a bird of the Muscicapidae in Malaysia. Bartlett & Greiner (1986) transferred to Pelecitus two filarial species parasitic in mammals, previously placed in the genus Dirofilaria: Pelecitus roemeri (von Linstow, 1905) in the knee region of Macropus giganteus Shaw (Macropodidae) in Australia and Pelecitus scapiceps (Leidy, 1886) in the ankle region of Sylvilagus floridanus (Allen) (Leporidae) in North America. Later, P. meridionaleporinus was described from the subcutaneous tissue at the ears of L. flavigularis in Mexico (Jim enez- Ruiz et al., 2004). Pelecitus roemeri can be clearly distinguished from the new species in that its females (23-110 mm long; from M. giganteus) are larger, its spicules are not strongly sclerotized and the left blade is slender (Bartlett & Greiner, 1986;Jim enez-Ruiz et al., 2004;Spratt, 2011). Contrary to P. copsychi n. sp., in P. scapiceps the pre-oesophageal ring is not readily apparent, the oesophagus is indistinctly divided, and the vulva is situated posterior to the oesophago-intestinal junction. In addition, females of P. scapiceps are larger (25-30 mm long) (Sonin, 1975;Bartlett & Greiner, 1986;Jim enez-Ruiz et al., 2004). Pelecitus meridionaleporinus differs from the present species in having larger females (12.5-28 mm long) and Based on the morphological and morphometric differences outlined above, we consider the present specimens as separate from their three congeners in Indomalaya and Australia as well as from the remaining 17 congeners in avian and mammalian hosts worldwide (Supplementary Table S1).
Molecular identification
A p-distance threshold has to be established to conclude if two sequences represent the same or different species. The optimum threshold (OT) is the p-distance value minimizing the false-positive and falsenegative errors in the intraspecific and interspecific assignments (Ferri et al., 2009). Owing to the lack of intraspecific data in the present dataset, the OT could not be estimated. However, Ferri et al. (2009) previously estimated an OT for the cox1 marker of the Onchocercidae. Their p-distance threshold, set at 4.8%, was used in the present study. The mean interspecific distance established for the present dataset was 14.67% (S.E. ¼ 2.15%; range ¼ 2.76-23.03%), which falls within the range of other similar studies (Ferri et al., 2009;Lefoulon et al., 2017).
The calculated p-distance for cox1 gene sequences between P. copsychi n. sp. and closely related species was 11.2% for F. candezei, 11.2% for L. loa and 14.1% for P. fulicaeatrae. The genetic variation between the present two specimens (ID nos. 168-4 and 268-5) obtained from the swollen footpad of the host's left leg was 0.4%. In Onchocerca spp., cox1 intraspecific genetic distance is lower than 2% (Lefoulon et al., 2017). Therefore, our molecular results corroborate that P. copsychi n. sp. is distinct from P. fulicaeatrae at the species level, whereas the genetic variation between the two specimens from C. malabaricus corresponds to intraspecific differences.
Molecular phylogeny
Overall, the concatenated phylogeny was well resolved and strongly supported. The Bayesian inference analysis resulted in a tree with five monophyletic Onchocercidae clades (ONC1-5) as previously proposed by Lefoulon et al. (2015). Most genera included in this study appeared as monophyletic (Fig. 3). The subfamily Dirofilariinae was divided into two clades: the genus Dirofilaria was placed in ONC3, whereas Pelecitus, including P. copsychi n. sp., Foleyella Seurat, 1917 andLoa Stiles, 1905 were placed in ONC5 (Fig. 3). Pelecitus is the only genus in this clade whose members parasitize birds and mammals, while species of Foleyella infect squamates and L. loa only infects humans (Bartlett, 1986;Bain et al., 1998). Notably, the genus Pelecitus presented as paraphyletic in that P. copsychi n. sp. together with F. candezei formed a well-supported clade as sister group to P. fulicaeatra within the Bayesian tree (Fig. 3). Affiliations within the ML phylogeny were similar to those in the Bayesian tree, although deeper nodes were less strongly supported (Supplementary Figure S1).
Wolbachia detection
Following immunohistological staining, Wolbachia endosymbionts could neither be detected in the genital system nor in the lateral chords of sections of a female P. copsychi n. sp. Similarly, nested PCR analysis failed to detect the presence of Wolbachia in the two specimens screened. Bartlett (1986) and Bartlett & Anderson (1987b) stated that although species of Pelecitus from birds exhibit a close morphological resemblance to Foleyella from squamates, many species of Pelecitus have a corkscrew-shaped body, whereas all adults of Foleyella spp. are straight or gently curved. Consequently, based on the corkscrew-shaped body in adults of both sexes as well as the predilection site in the host and the host taxon itself, we assigned the present specimens to Pelecitus. Bain et al. (1993) suggested that the anterior position of the vulva in Onchocerca ramachandrini Bain, Wahl & Renz, 1993 from Phacochoerus africanus (Gmelin) (Suidae: Phacochoerinae) in the Afrotropical realm represented one of the ancestral characteristics within members of the genus Onchocerca Diesing, 1841, while a short, undivided oesophagus was considered an evolved character. In the Oswaldofilariinae Chabaud & Choquet, 1953, a long oesophagus was viewed as an ancestral characteristic (Chabaud & Bain, 1994). If these hypotheses are correct, we propose that P. copsychi n. sp. has some ancestral characteristics in comparison to its congeners P. ceylonensis, P. galli and P. bartneri from Indomalaya and Australia. The position of the vulva is anterior in P. copsychi n. sp., situated at the level of the anterior half of the oesophagus, not the posterior half, and the oesophagus is longer. In addition, the oesophagus is divided in the new species (as well as in P. bartneri), but undivided in P. ceylonensis and P. galli (Table 1).
Discussion
With regard to the geographical distribution of Pelecitus spp. from birds, many species have been recorded in Africa and South America and only three species have been reported to date from Indomalaya and Australia. In the present study, we describe P. copsychi n. sp. from C. malabaricus in the primary forest of Pahang, Peninsular Malaysia. This bird has not been listed as host of previously described species of Pelecitus (see Bartlett & Greiner, 1986;Spratt, 2011;Supplementary Copsychus malabaricus is distributed in the southern Thai-Malay Peninsula (Rasmussen & Anderson, 2005). Although P. copsychi n. sp. was discovered in close geographical vicinity to P. galli, its host, C. malabaricus, belongs to the order Passeriformes, whereas the host of P. galli, G. g. spadiceus, belongs to the order Galliformes (Dissanaike & Fernando, 1974).
Microfilariae were not found in the blood smears of C. malabaricus, suggesting skin-inhabiting microfilariae. According to Bartlett & Anderson (1987b), the skin-inhabiting microfilariae of P. fulicaeatrae are extraordinarily rare, and skin-inhabiting microfilariae have not previously been reported for any of the more than 140 known species of avian filariae. In future studies, skin snips should be taken from birds to test this hypothesis.
Regarding zoonotic infections, Bain et al. (2011) suggested that an intraocular worm from a patient in Brazil belonged to the genus Pelecitus. Similarly, an intraocular worm from a patient in Colombia was originally identified as Loaina sp. (Botero et al., 1984), but Bartlett & Greiner (1986) considered it more likely that the worm belonged to a species of Pelecitus parasitic in birds. Bain et al. (2011) supported their opinion. While we conclude that Pelecitus spp. of avian origin may be of zoonotic importance in South America, evidence of the zoonotic potential of the four Pelecitus spp. (including P. copsychi n. sp.) from avian hosts in Indomalaya and Australia has yet to be found. Bartlett & Greiner (1986) suggested that Pelecitus spp. of mammals represent "capture" of avian filariae by mammals. In other words, Pelecitus spp. in mammals could have originated from avian filariae through host-switching events facilitated by vectors.
The vector of P. ceylonensis in birds is a mosquito, namely Coquillettidia crassipes (van der Wulp) (syn. Mansonia crassipes (van der Wulp)) (Diptera) (Dissanaike, 1967), and development of P. fulicaeatrae was recorded in the chewing louse Pseudomenopon pilosum (Scopoli) (Mallophaga) (Bartlett & Anderson, 1987b). In mammalian hosts, the vectors of P. scapiceps are mosquitoes and those of P. roemeri tabanids (Highby, 1943;Spratt, 1972). Therefore, Pelecitus species possess the most heterogeneous vector range within the Onchocercidae. Although we did not find any potential vectors for P. copsychi n. sp. in the present study, we speculate that, like its congeners, the new species is transmitted by haematophagous arthropods.
The division of the subfamily Dirofilariinae is not only wellsupported from a molecular standpoint (Lefoulon et al., 2015;this paper), but can also be observed in certain biological particularities. First, the third moult occurs as early as day 2-3 post-inoculation in Dirofilaria spp. (similar to Onchocerca spp.), but at a later stage, approximately one week post-inoculation, in the remaining genera of this subfamily (Bain et al., 1998). Secondly, the genus Dirofilaria shares morphological characteristics with the genus Onchocerca: buccal capsule normally developed in infective larvae but reduced in adults; a cylindrical tail with rounded extremity and tiny caudal lappets in infective larvae (Bain et al., 2008).
Based on the redescriptions of F. candezei and Foleyella furcata (von Linstow, 1899), Bartlett (1986) suggested that the close morphological resemblance between Foleyella and Pelecitus indicates their close phylogenetic affinities. The present phylogenetic analysis revealed Pelecitus as Fig. 3. Clades of the Onchocercidae (ONC1-5) based on partitioned concatenated datasets of 12S rDNA, cox1, rbp1, hsp70, myoHC, 18S rDNA and 28S rDNA sequences. The total length of the dataset is c.3979 bp. Fifty onchocercid sequences (representing 49 species) were analysed. Filaria latala and Protospirura muricola were used as the outgroups. The topology was inferred using Bayesian inference on two runs of five million generations, with the first 25% of the trees removed as "burn-in". The onchocercid subfamilies are indicated by colour: blue for Onchocercinae, dark green for Dirofilariinae, purple for Splendidofilariinae, pale green for Setariinae, yellow for Waltonellinae, orange for Icosiellinae and red for Oswaldofilariinae. The red triangles indicate the sequences generated in this study. paraphyletic (Fig. 3). Two hypotheses may be put forward. Pelecitus may have a complex evolutionary history, possibly owing to its worldwide geographical distribution and a broad host spectrum at genus level (11 orders, representing more than half of the avian orders and two mammalian families) (Bartlett & Greiner, 1986;Jim enez-Ruiz et al., 2004). Alternatively, the current limited set of molecular data (two species out of 21 known species, including P. copsychi n. sp.) might not be inclusive enough to support reliable conclusions about the phylogenetic relationships of this genus. Adding molecular data for species of Pelecitus representing a wider host spectrum and geographical range may in future provide clarification. In the present phylogeny, Pelecitus formed a clade with Foleyella and Loa (second clade of ONC5). This clade presents the most heterogeneous host range within all onchocercid clades, comprising reptiles, birds and mammals.
To date, five species of avian filariae have been tested for Wolbachia endosymbionts and have been found Wolbachia-free: Chandlerella quiscali (Linstow, 1904), A. alessandroi and C. pavlovskyi in the Splendidofilariinae Chabaud & Choquet, 1953 well as P. fulicaeatrae and P. copsychi n. sp. in the Dirofilariinae (McNulty et al., 2012;Lefoulon et al., 2016; this study). One might hypothesize that avian filariae in general are devoid of Wolbachia. However, compared to the total number of filarial species infecting birds (c.170 according to Bartlett (2008)), the number of tested species is still too limited to support a reliable conclusion. In addition, L. loa and F. candezei do not harbour any Wolbachia endosymbionts either (McGarry et al., 2003;Lefoulon et al., 2016), suggesting that this entire sub-clade within ONC5 may be free of Wolbachia ( Fig. 3; Supplementary Figure S1).
Conclusions
Pelecitus copsychi was described as a new species from C. malabaricus in Peninsular Malaysia, integrating morphological as well as molecular characteristics. The filariae occurred free in the footpad of the host and caused swelling at the site of infection. Copsychus malabaricus represents a new host record for the genus Pelecitus. Phylogenetic analysis based on seven genes (two mitochondrial genes, 12S rDNA and cox1; and five nuclear genes, 18S rDNA, 28S rDNA, MyoHC, rbp1 and hsp70), revealed the new species as closely related to P. fulicaeatrae from birds and F. candezei from squamates in Africa. However, cox1 analysis supported the new species as being distinct from P. fulicaeatrae at the species level. In the Bayesian and maximum-likelihood inferences, P. copsychi n. sp. clustered with other members of the Dirofilariinae as well as some genera of the Onchocercinae and Splendidofilariinae in a well-supported clade named ONC5, sister to all other taxa evaluated within the Onchocercidae (Lefoulon et al., 2015). The present study corroborates the division of the Dirofilariinae into two distinct groups, linking Dirofilaria spp. on one side and P. fulicaeatrae, P. copsychi n. sp., F. candezei and L. loa on the other side.
Funding
This study was supported by the Ministry of Higher Education, Malaysia (FRGS FP020-2012A).
Ethical approval
Culling of animals and all experimental procedures were carried out in strict compliance with the policy and protocols approved by the Institutional Animal Care and Use Committee, Universiti Malaya, Kuala Lumpur, Malaysia (protocol No. S/15102018/31082018-02/R). The surveys were carried out in accordance with the conservation and control policies of the Department of Wildlife, Malaysia.
Declaration of competing interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. | 2022-02-01T16:10:34.378Z | 2022-01-01T00:00:00.000 | {
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264835871 | pes2o/s2orc | v3-fos-license | THE STUDY OF COMMUNICATIVE INTELLECTUALITY PLACE / ROLE IN THE EXPANSION OF GENERAL DOMAIN FROM THE PERSPECTIVE OF MODERN THEORISTS OF COMMUNICATION
The main idea of the lack of a master and general plan concerning the status of rationality and its evolution was shaped as a communicative rationality in communication science. The communicative place rationality in the public domain from the perspective of modernity theorists is the basic question in this field. Procedure of the research is qualitative research method with qualitative analysis of Max Weber. The first step of the research is an overview of research topic, objectives, research methods and Statistical Society. The next step is a deep study in the evolution of history of ancient Greece, during the development of Christianity and the explanation of votes in the Christian era, the Enlightenment and the intellectual revolution. Also, entries on topics such as the definition of modernism and modernity that define the modern scientific achievements and the consequences of modernity and its crisis on modernity and rationality is studied in this research. Finally, the communicative rationality and rationalization of the development and differentiation and achieve communicative rationality is deliberated. This section is fed mostly by Habermas's ideas and thoughts.
INTRODUCTION
Revival/Renaissance Renaissance in English, or the era of Rebirth was an important cultural movement that became the starter/starting point of a scientific revolution, religious reforms in addition to an era of artistic advancement in Europe.The era of Rebirth is a transit period in the middle ages and the new era.For the first time, the word Renaissance was used by the French people in the 16 th century.14 th century is known as the beginning of Rebirth period in the north Italy.This movement also covered the north of Europe during 15 th century.Renaissance is a 300 years old evolution that was begun from the Florence and ended during the Enlightenment period in the Europe.
The Renaissance was started from Italy in 1300 and spread in the whole Europe during three centuries.It is very rare that a diverse and historical event could occur in such a short period.While these centuries are full of fundamental changes and great activities, the today's world is the aftermath of such activities, since the Renaissance founded the economical, political, artistic and scientific pillars of present civilizations in the Europe (Koric, 2005).
The knowledge and the art had brought the great advancements into existence in the Italy of 15 th and 16 th centuries.This cultural revival has become famous as Renaissance (i.e.Rebirth).Many scholars, composers and philosophers emerged who looked on the world with the newer oculus/eyes inspiring by the genuine heritage of Rome and Greece.Painters had engaged in the study of human body and painted the human body organs with a realistic method.The Rulers ordered the great artistic works and buildings.These fresh beliefs suddenly expanded in the whole Europe.
Expression of communicative Views and beliefs during Renaissance
1. German Emanuel Kant (1724-1804): considers the characteristic of this era the desire of human being to know the reality.2. German Martin Luther : protested against the abuse/exploitation of church.We see the enlightenment of Protestant on its continuation.3. John Lock (1632Lock ( -1704)): one of the other pioneers/pathfinders of this revolution founded the intellectual foundation of enlightenment through his theories and writing works.In view of Lock, all human beings have the power of thinking and the intellect itself shows that all human beings enjoy similar natural rights for desiring the life freedom and proprietorship.4. Rene Descartes (1596-1650): was one of the most respectful supporters of intellect and used something similar to mathematical logic to find the reality.A fresh wave of thinkers and writers emerged during the middle of the 18 th century whose viewpoint was different with the older speakers of enlightenment movement.These modern social critics had no further delusion about intellect.They considered the cause of many social complications in the unlimited relying on intellect; several of the influential thinkers and writers had role against the intellect whose most powerful is the Jan Jack Russo.
The enlightenment period despite its abundant achievements passed and during the next century encouraged the criticisms in the context of Votes rights and world opinion.Many of the interpreters have blamed the philosophers that had caused the activating of rioting force that demolished the traditional sources of power, stability and discipline.Some of the critics proclaim that the enlightenment caused the existence of hopelessness, being without goal and solitude that is the characteristic of modern era.They argue that the enlightenment has an unlimited emphasis on the individual rights in place of the individual responsibilities and social obligations.Besides this, the philosophers were blamed for weakening the religion power and trust in God without showing any substitute for responding to the spiritual/religious needs of most people.The heritage of enlightenment has also made the environment supporters unhappy who say the emphasis on the advancement and intellect in the effective materialistic approach has been effective in encountering the nature world.(Machiavelli book and the Renaissance thought by Jahanbagloo (1991).
INTELLECTUAL REVOLUTION OF DESCARTES
One of the intellectual revolutions was started by Descartes; Descartes replaced the human intellect in place of sacred book and tradition of Pope, church and ruler.Descartes created a great topic with this step.Western Europe eliminated the philosophy of Descartes and the era of "Bright and distinctive thoughts" started, everything was measured and weighed by the criteria of intellect; even the contents of sacred bible in the belief era gave its place to the era of intellect.The method of Descartes for research on nature was inferential and suppositional.
Modernity
Until now, diverse definitions have been made of modernity and the extracts of word "modern", however these definitions have in no sense, eliminated the complexity from the actual concept of these words, or even increased the intricacy of it.The word modern that is equally used in all European languages and many of the other languages including Persian, has its root in Latin word "Modernus" that is extracted from the word "Modo."In the Latin language, the word 'Modo' means, recently, lately and very near past.A few of the history writers suggest that, the word Moderni, was used by Romans at the end of 5 th century about values and the new distrustful beliefs; the words which were in comparison with the ancient accepted beliefs which were specified by the word "Antiqui." Modernity that was considered the intellectualism period in the world of intuition/observation and disconnected from the invisible world with the exploitation goal, scientism, materialism and the thought of linear advancement, has transformed the life structure, human relationship as well as the comprehension of the modern man.(Ahmadi, 1998)
Study of modernity concept from Jurgen Habermas viewpoint
Habermas in the definition of new and modern word emphasizes on its relation with the past and knows the traditional past and the awareness resulted from living in the new and modern period to an ancient pattern in the past and the necessity to transit from it as well as applying the modern pattern: "the term of modern with different meanings and concepts, repeatedly expresses the awareness with an era or a period that connects itself with traditional past so that could regard itself the result of transit from the old to the new…in other words, the term of modern emerged and reemerged accurately during a period in the Europe that in which the awareness in relation with modern era took shape through the revival of relationship with the ancient people (Habermas (2005), Tadin (2005), Nozari ( 2002)).
Habermas considers the romantic soul of 19 th century resulted from the core of idealized middle ages that intensified the modernity awareness and turned it to a form of radical awareness.However, he considers this comparison between tradition and present as an abstract comparison.
In the words of Habermas, the intellect resulted from the enlightenment lies in three domains: first domain is the field of science whose logic is the instrumental intellect not the communicative intellect.Second field is the domain of ethics whose reliability is taken from the norm while the third domain relates to the art that is fed by the beauty.From the viewpoint of enlightenment thinkers, these three domains can cause the freedom of humankind and reduce the depth and the extent of existing problems and this is a defendable project.In the view of Habermas, being in line with the specializing of these three domains, they detached from everyday fields.For instance, in the domain of art, the art not with goodness but interpreted with the reliability of a climax inside its own or art for art only.Therefore, for liberation, the mentioned three domains should coordinate with each other and the modernity has the power and ability to be the caller of liberation (Habermas, 1998).
Intellectuality and Modernity
After passing the middle ages in Europe and entering into modernity, in general, an idea was discussed by Descartes as the main theory of modernity and this was the idea that in contrary to the middle ages that was the main factor tradition and introducer of world to the mankind, after that the man itself became the introducer of world and nature as well as the concepts around the western society.This idea became the background of all evolutions of this era in the west.But this was not the only stroke to the modernity thought but after a while the realities of the world and the modernistic look to the world was also challenged by Marx.
Modernity became the main discussion of the sociology.The previous social theorists despite that had the more complex and more impartial perceptions on human situation.They in general, had less absorbed by the inevitable optimistic views in the human advancement through intellectual rationalism (Mohammadi (2001) and ( 2003)).
Modernization as the intellectuality
During the Renaissance the initial signs of modernity revealed a concept of extended rationalist 'Hmanism'.The goal was to increase the tolerance and intellectuality in all aspects and to achieve this goal they supported a necessary opposition for role playing.The tradition could not further be a justification for the human acts.The human world must be considered in the center of focus with certainty and the main objective should be the speculation, the change of religious state and speculating on the reality of Lord and his will.The needs, powers and the human interests should be the base and foundation of all values, and the social life should be changed based on these instances.Therefore, the human life should be organized only through logic: the movement should be towards a society where the rule of intellect and logic should replace the rule of Lord.Limiting the rationalization as the instrument to provide more satisfaction should be seen in the human life affairs.The Renaissance struggled against the absolute affairs of middle ages called the absolute skepticism and encouraged the doubt and misgiving.The fundamentals and the structures of mental proclaim that had advanced in the enlightenment process, its theorist was Marx Webber, who objective was to describe and interpret the rationalization in the communities of Europe which had also transferred to the other parts of the world.Weber rejected the beliefs of the theorists who saw the growth of rationalism as the inevitable result of a direct and global process of social evolution (Pooladi 2004).
CONCEPT OF INTELLECTUALITY IN THE THOUGHTS OF MAX WEBER
The targeted intellectuality mainly by applying the best possible under possession tool follows its fixed objectives.Webber believes that this type of intellectuality has dominated the western culture and the valued intellectuality has lost its color.However, regarding these two types of intellectualities does not take one sided stand.Therefore, we cannot recognize Webber as the herald thinker of intellectuality and intellectualism.
Webber with a historical viewpoint tries to analyze the nature of modern social life and notices in this framework that a type of being intellectual in the sense of very instrumental intellectuality has influenced the direction and dimension of human society during the history span especially in the modern era, specifically in the West where the intellectuality could come in to existence in this form of foundations such as modern state (discussed in the framework of Bureaucracy), capitalism economy and in the formation of modern rights.
Roger track rightly believes that the intellectuality problem, challenges the scholars of social sciences at two levels, the intellectuality manner of under study individuals is an issue, while the intellectuality of the scholars of social sciences is another problem (Cooke, 1994).
If we would have comprehended this separation in right way, the intellectuality of first type dates back to the intellectuality of the actions of actors while the second type of intellectuality to its standard, as scientist accept and love the scientism of social sciences.This is an important separation; of course it was also under consider in the discussion of Kent and Part, and presented their theories on this base.However, later on we would focus a serious attention to it (Habermas (2005)).
INTELLECTUALITY
This concept has had abundant applications, has been used in different areas.That is why, it has distinctive meanings.Perhaps by inducting the areas in which the word of intellectuality has been used, a comprehensive meaning could be taken in to consideration for it.This comprehensive meaning could be considered as the complete following of correct argumentation.It is clear that arguing on one thing has different ways that is why the intellectuality resulting from different types of argument would also be different.
Intellectuality is divided into theoretical and practical intellectualities in an initial classification.The theoretical intellectuality discusses the beliefs and world sighting while the practical intellectuality considers the actions and behaviors.Some have defined these two in this way: the theoretical intellectuality expresses more accurate mastery or domination on reality through the abstract concepts while the practical intellectuality means the probably more accurate calculation of the means to achieve a goal.
However, the ethical intellectuality of man is called by justice and equality and asks him not to oppress the others in pursuing ones goal and don't sacrifice their rights for him.Summarily, this intellectuality for pursuing the personal interests specifies the framework that is the very boundary of ethical and moral values.
Intellectuality's concept and being Normative
A normative definition is made in defining the intellectuality.Intellectuality is an abstract concept extracted from the adjective of intellectual.We are compelled to investigate this trait to define intellectuality.
Instrumental Intellectuality
Dominant thought in the framework of intellectuality based on the theory of intellectual selection focuses and emphasizes on the instrumental intellectuality and the result logic.Based on this approach, the actors think about the result of their selection and deal with the decision making by analyzing cost and profit.
The instrumental intellect orders that the best and the cheapest tool should be applied to achieve the most profitable objectives.If the selection of optimal option in the sense of absolute intellectuality is not available, the choosing of a cheaper option is equal to the inevitable limited intellectuality.The criterion of intellectuality is the success rate in acquiring the interests/profits and developing the desired results.This type of intellectuality is called instrumental intellectuality.
Moral Intellectuality
Other type of intellectuality is the moral intellectuality.The moral intellectuality is necessary with the proportion logic and focused on the specification and determination of duty or obligation as well as decision making and the appropriate action to it.
Communicative Intellectuality
Other type of intellectuality is known as the communicative intellectuality.As per the view of Habermas the relation through tongue necessarily needs the posing of reliability claim specifically the reality of truth fact whose situation at the time of views difference could only be solved through conversation/discussion.Besides that Habermas believes that the native of a language completely recognizes the conditions of such conversation that brings actually the positive results and he has explained these conditions in terms of attributes of justifying and idealistic traits of speech.The communicative intellectuality, implies on this point that the man/humankind has this ability that he engages in protest in the conditions close to this idealistic situation ('discourse' as per Habermas term) and with the goal of access to agreement.
Habermas relies on the concept of intellectuality so that to describe this point whatever is shown by the democratic forms of social organization, is more beyond the very priorities in the cultural and political tradition.In his view, the speech action cannot be even understood without adopting the stand regarding reliability claim that he discusses and this stance itself prepares for the conversation free from limitation that situation specifies (this) claim.Therefore, the social and political arrangements can be criticized, because as per the outlook of Habermass, that reaching the agreement is either a goal that is the personal object for human tongue.A same philosophical program has also been discussed by Karl Otto Opal; he more emphasizes on the glorification traits of protest.
Habermass makes clear that the participants in the communicative action should see the statements discussed during conversation from the viewpoint of their reliability, and also should have full cognition in the context of appropriate ways to solve the difference instances on the claim of credibility as well as in the context of the conditions (that is always against reality) in which the following up of such methods would actually give the correct result.For instance, the pursuance to imposing the hidden pressure makes previously obtained agreement unreliable, this cognition becomes mobilized.The comparison base between instrumental and communicative intellectuality that indicates the endeavor of Habermas in the direction of treating this situation is in the philosophical anthropology which has been presented systematically in the 'cognition and the human interests' (Habermas 1981).Habermas suggests here that every society for its reproduction should be powerful on the exchange of generator/producer with nature (in the work form) in addition to the communicative coordination of the collective activities.Work creates the concepts that express the 'technical interest' to instrumental control, while the need to agreement creates the framework of distinctive category and practical interest to the hermeneutic understanding (Habermas, 1988).
Habermas replaces the theory of discoursing reality with the theory of conformation reality.While, the second theory was challenged by the Cartesian science of criticism's subjectivity and the text free intellectuality.Habermas claims that the first theory which refreshes itself with every communicative action can save us from the morass of relativism.
Habermas applies the interpretation of general domain in attributing to the social area in which the individuals produce the stances and valued and normative orientations through mutual understanding and argumentation based on thinking and in the conditions free from every type of pressure, force and under equal conditions for all sides of participants of behavior collection, stands and valued as well as normative orientations.
What has caused the attracting of Habermas' view to the topic of general domain has been the importance of this title as the criticism base of society, based on democratic principle.The general domain is the area in which the individuals get together to participate in the open and free discussions.In view of Habermas, the general domain in the accurate sense of words is an area that no limit and constraint would have been enacted on its activity.The general domain is the intellectual, fact finding and reality making domain of the society.
Habermas is following the revival of general domain by creating and expanding the theory of communicative action.The intellect discussion (conversation) free of domination and the linguistic damages and agreement and consensus is the very local type that is appropriate for general domain.In the view of Habermas, the theory of communicative action can be applied as the fundament of general principle.
The communicative action has a social situation.In the communicative action the agent/operant person has no weapon except logical argument.Human beings can access the agreement and correlation more freely and easily without direct distress and indirectly the necessary material problems in a process of view exchanging.The objective of such social action is the cognition of all and accepting the same realities.This domain in fact is a social ideal.
Answers are not ready in priory but the people select their ways by view exchanging and criticizing them.In the communicative action, the participants exceed their cooperation and there are no more mathematically dry calculations as well as the pre-existing pressure but people try to select the ways for agreement through discussion and conversation.This action is liberating intrinsically, because it accompanies high actions.This liberation could materialize in a bad and ground in which the self recognition would be possible through discourse.(Habermas, (1976)) Habermas believes that the theory of being intellectual of Weber enjoys significance to understand the modernity that is the fundamental issue.We follow the modernity issue/problem through the works of Max Weber since his 'errors/ mistakes' are considered the visualization of intellectual and practical indigence/helplessness in facing our own period.
On the other hand, we are fully satisfied that transit from the traditional societies to our present societies is equal to liberate from magic and sorcery, and has been from several types of sacred dread that provided the arrangements to investigate the beliefs, unhappy end of Galileo as well as the Sorcerers of Salem, this advancement is known as intellectual progress alongside the science evolution.Weber names this advancement the process of 'incantation and de-hallucination.'On the other hand, when we look on our own period with keen eyes, undoubtedly we see everything completely different.All of a sudden the advancement changes to the growth of independent half of science and technology, takes control of our reign and crosses us through a path that Habermas, the process that pulls us directly to the 'iron cage' of recent capitalism.
The problem lies here; Habermas tries to give a compromise between two process of sorcery and dehallucination in a single theory of modernity and the modernization.To do this in appropriate way, it is necessary to reconstruct the theory of Weber, the reconstruction that could analyze the theory in separate components and collect it in a modern form to reach the goal the theory accepts for itself in a complete way.In the modern sociology, four types of intellectuality have been introduced by model making of Max Weber's theory.These four species as per Kleberg are as follows: theoretical, practical, intrinsic and external (structural).
In contrary, Habermas presented the new classification of intellectuality.He suggests that we face two categories of intellectuality, first the instrumental intellectuality that in his words, all endeavors of Weber had been passed in explaining such type of intellectuality.Other, is the cultural-valued intellectuality focused on the goals of actions.The cultural-valued intellectuality watches the correction of communicative actions; this process is possible through independent and logical discourse (Habermas, 1988).
Habermas identified two types of intellectuality in the Weber works: one the cultural-valued intellectuality and the other, official or instrumental intellectuality.Habermas believed that the cultural-valued intellectuality remained incomplete in the thoughts of Weber; however, the instrumental intellectuality has been much expanded in the theories of state, economy and Bureaucracy.Habermas tries to explain the first process of intellectuality.At the same time, he sees a dialectical relationship between these two processes of intellectuality.
The valued cultural intellectuality lies in the center of special type of action that is interpreted as communicative action by Habermas.In the communicative action of discourse or the speech relationship that is accomplished by means of tongue, identified as the characteristic of intellectual concept among the actors.Habermas before introducing the communicative action explains two types of actions i.e. 'instrumental action' and 'strategic action' in a size that helps the expression of communicative action.These two actions lie under the umbrella of instrumental intellectuality that follows a reasonable instrument to reach the given objective.Though, the instrumental action is intellectual but considered as individual that resembles the 'practical intellectuality.'Contrarily, the action is a collective strategy that is followed by social structure or organization.Bureaucracy or the structure of modern society is administered based on the strategic action that is the very example of high instrumental intellectuality.Habermas, in contrary to these two, puts a communicative action that is social firstly; secondly it is intellectual in terms of goal and end; because, the goal choosing in this action takes place based on discussion and free discourse, as well as intellectualism that has been freed from all types of structural and political domination.
Contrary to the reasonable and targeted action whose main orientation is formed by accessing the opportunistic success, the communicative action and communicative intellectuality have the orientation of understanding.Therefore, the actions of the related persons, are coordinated not through opportunistic success calculations, but through the comprehensible actions.So, the engaged people in communicative action are not restricted to personal success but follow their goals under the conditions in which they could coordinate their action plans based on a definition of 'common success.'Habermas defines the action and the communicative action in this way: the communicative action implies the interaction of at least two factors which are able to speak and establish the personal relations.He names the Webry rationalism as instrumental intellectuality.
This could be said on the viewpoints difference of these two: The valued intellectuality of Habermas is focused on the correction and reconstruction of the goals and societal values; while, the instrumental intellectuality is focused on optimizing the methods and the means.
The Webry intellectuality often notices the individual actors as well as the sharp action of them, while the valued intellectuality also emphasizes on the social and structural dimension.In the instrumental intellectuality only the reasonability and being targeting of action has importance while in the valued intellectuality, the communicative action and the distorter/falsifier factor is also under consideration.In view of Habermas the intellectuality is a norms giving instrument, obligatory and arising from the social system especially in the form of formal intellectuality that is imposed on the actors.While the intellectuality is a value based on free discourse and far from domination and its result is the common and collective agreement.(Adorno and Horkheimer, 1944).
FALL OF GENERAL DOMAIN AND COMMUNICATIVE INTELLECTUALITY
However, the Habermas refers to the attenuation manner of general domain and believes that the state interference, part politics and the manipulation of mass media weakens the general domain and the general domain and the communicative intellectuality changes to an ideological element.Habermas believes that the cause of the genuine democratic debate's fall is considered a role played by the monopoly mass media in keeping the general public unaware and optimist.
If the general domain goes under the domination and the control of a number of organizations and the powerful giant establishments, this domain would change to a mean and instrument for manipulating the general interests and the minds and each and every particular beneficiary group by accessing the mass media means, tries to demonstrate its specific interest as the general interest that this confusing effect taking is considered as one of the traits of autocrat and authoritative governments which easily turn the people to the tractable herds.Therefore, the interference in general domain is one of the pillars of power seeking governments.(Habermas, 1988).
Habermas under Critics
From the very beginning of inserting and resulting of communicative intellectuality theory of Habermas, this theory and its theorist went under the fire of a number of critics that the following instances could be named in this regard: 1.The moral theory of Habermas in the real sense is a 'monologue.'The reason is that the mentioned credibility giving conversation the conversation moral relies on is an idealistic dialogue, not a real conversation.In the real life, the idealistic situation of speech as well as the speech of Habermas never materialized completely, and possibly would never be materialized in the future, even if its reason is its time limitation.Besides that, the group of people who are exposed to the effect taking of a norm they must agree upon it, possibly includes the people who have not still born in this world.Therefore, it is always possible for Habermas that he does not accept the agreement conditions that have been obtained in the real dialogue.If we are set to put the dialogue moral in practice, the moral factor can only conclude that which type of agreement is achieved in the idealistic dialogue.Such thing is only the arrangement of discovering and exploring that could only be used in the memory of a scholar i.e. in the monologue form (Nozari, 2002).2. Habermas uses the psychoanalysis as a model to express the concept of liberating social science; however the treatment relation is different from that of actual social condition.In the treatment relation, on one side the voluntary relationship obeys the other, but in the social contradictions we always encounter the situation of two sided resistance.3.This shaping of Habermas is of limited significance from the sociological viewpoint.Because it not sufficiently general and has its roots in the awareness philosophy more than needed and has been tied with a picture of an individual and sole speaker and listener and does not include the oral society.4. Finally, whether the communicative intellectuality of Habermas is beyond the instrumental intellectuality, i.e. not an appropriate selection as means-goal.Finally, the dialogue is also a means for achieving a given objective.5.The shaping or formulating of intellect in the world of humanitarian sciences relying on the using methods of empirical science is included in the discussions that have been under serious and grave critics of Habermas.On one hand, passing through the instrumental intellectuality positivistic and targeted forms, towards the communicative and conceptual intellectuality that is based on the sound and liberating human actions.On the other hand, the exit or the Go out from the degeneracy (iron cage) of capitalism's intellectuality is considered as the theoretical approach of this political thinker/philosopher. 6. Opal prefers the term of "lofty pragmatics" on "global pragmatism" of Habermas for the general theory of communicative skill.The main differences between Opal and Habermas, dates back to their attitude on the possibility of "Letzt begrundung" (last argument) for moral and combative/Jihadist stance.Apel (1990) in a critical article stated that Habermas mistakes who underestimates the (lofty difference) between the reconstructions of communicative skill that has the ability of empirical test and philosophical deliberation, the deliberation that reveals the unconditional credibility of some basic presumption of protest.
Summarily and generally, we have compiled the theories inference of the theorists regarding the intellectuality in the form of a progressive table.communicative intellectuality is that in which the goal of subject is related to the required goal of receiver/acceptor and believes that the intellectual action is an action that should enjoy similar and same meaning in terms of objectivity as well as intellectuality and mind. | 2019-07-30T17:09:57.914Z | 2016-01-01T00:00:00.000 | {
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265004917 | pes2o/s2orc | v3-fos-license | Social and environmental features in domestic and international minerals resources classification systems
This paper is aimed at a comparison of Ukrainian and international resource classification systems’ fundamental features. As a result, the most common and different features were outlined. The importance of accounting for the social and environmental viability of projects is determined. This study highlights the significance of the harmonization of domestic classification with international standards.
Introduction
All known international standards for the assessment of reserves and resources, as well as domestic regulatory documents, contain object classifications according to important geological and industrial features.These systematizations don't appear by chance, and as a rule, reflect the traditions and stages of exploration and evaluation of mineral deposits in a particular region.
The classification of mineral reserves and resources of the State Subsoil Fund establishes the principles for calculating, geological and economic assessment and state accounting of mineral reserves that are common for the State Subsoil Fund of Ukraine according to the level of their industrial significance and the degree of geological and feasibility study, the conditions that determine the readiness of explored mineral deposits for industrial development, as well as the basic principles for quantifying mineral resources [1].The practice of using this classification systematizes mineral reserves and resources according to certain levels of industrial significance and the degree of technical, economic, and geological study.This classification contains distributable classes that are identified using an international three-order numeric code.Today our classification is harmonized with the United Nations Framework Classification for Resources (UNFC) 2009 version despite the fact that there is already an updated version of 2019 [2].
The relevance of this study is related to the integration of domestic subsoil use into European industries, as well as into the global systems for investing in mining facilities.Thus, the domestic mineral resource base takes into account many minerals from the list of critical and strategic ones for such countries as the United States of America, Canada, Japan, Australia, and the European Union.Understanding the availability and maturity of our deposits will accelerate their investment and development.Understanding the requirements of international standards 1254 (2023) 012098 IOP Publishing doi:10.1088/1755-1315/1254/1/012098 2 for the exploration of deposits will allow us to study quickly and efficiently own objects and select interesting foreign objects.
The analysis of classification systems allows us to determine directions for further changes and harmonization of the national evaluation system.Until recently, social and environmental factors were rarely taken into account when classifying mineral reserves and resources.Their importance has grown significantly over the past few years.Many projects have been delayed or canceled because they did not meet social or environmental expectations, even if they met all other requirements of quantity, quality, and profitability.The various factors involved in the classification of resources do not exist in isolation but operate in a complex manner.In particular, specific issues of property rights and use of the resource, licensing conditions, and other legal issues, which as a result also create economic conditions, can be affected by social and environmental risks.The delay caused by the resolution of these issues in connection with socio-environmental problems can have a significant impact on the economics of projects and even make them economically unprofitable [3].
Results
Until recently, social and environmental factors have rarely been considered in the classification of natural resources.Their importance has grown considerably in the last few years.Many projects have been delayed or canceled because they failed to meet social or environmental expectations, even though they met all other requirements.The various factors involved in resource classification do not exist in isolation, and the distinction between them is rarely black and white.The related issues of ownership, contract terms, legal, regulatory issues, and in some cases, financial conditions may be affected by social and environmental issues.A delay due to the resolution of these as a result of socio-environmental issues can have a significant impact on the economics of projects, even making them no longer economically viable.
Socio-environmental issues, typically described as a requirement for "social license" or "social license to operate" (SLO), have attracted a significant amount of interest and attention in recent years.A project cannot proceed unless the important social and environmental contingencies are resolved, typically described as obtaining an SLO.UNFC is a tool for effective management of national resource endowments needed for realizing the Sustainable Development Goals (SDGs).UNFC applies to energy and mineral resources; injection projects for the geological storage of CO 2 ; and anthropogenic resources such as secondary resources recycled from residues and wastes.UNFC aims to provide necessary specifications and guidelines for optimizing the management and development of resources, with positive impacts on society, the environment, local economies, and employment [3].
In 2022, the problems and risks of social licenses have become especially acute, which is especially noted by the forecasts of global audit companies.Table 1 lists the Top 10 business risks and opportunities for mining and metals.The risks of social licenses appeared in the lists of top risks in 2015, but until 2022 they did not rise above 5th place.
With Ernst and Young's (EY) data released at the end of 2022 [4], environmental, social, and corporate governance (ESG) remains a top risk and challenge for mining and metals companies in 2023.This issue is now widely included in corporate strategies due to its impact on almost all aspects of operations -productivity, logistics, economics and finance, sales markets, etc.Some of the biggest areas of ESG improvement are not new -ensuring diversity, equity, and inclusion still remain challenges, and mine closures and rehabilitation require longer-term, more strategic planning [4].
However, every year the demands on mining enterprises are becoming stricter and today, water resources management and biodiversity are fast becoming urgent priorities in the face of climate change.Stakeholders expect from companies a better assessment of risks and opportunities to minimize them with the help of transparent studies and guarantees for their .Stricter reporting will be critical if companies are to meet growing stakeholder expectations and avoid accusations of greenwashing.A bonus for companies that meet these requirements is significant competitive advantages -from access to capital and resources to obtaining a license to conduct activities and attracting highly qualified personnel [4].
Among the most significant for the extractive industry in the list of social and environmental factors were the following (in descending order): water resource management problems, compliance with the processes of decarbonization of production, reduction of the negative impact on climate change, environmental friendliness of production and volumes of emissions that are directly or indirectly related to enterprises, etc.
Classifications of mineral reserves and resources always reflect the most important features of mineral projects that determine their effectiveness.From the beginning, these were basic geological and mining technical characteristics, then economic and environmental factors.Now such a sign is environmental, social, and governance, which directly affect the possibility of project implementation.Next, we analyze existing system classifications to determine the direct and indirect ways in which these systems account for these ESG factors.
The development of international reporting standards for the assessment of mineral resources began at the end of the 20th century as a result of globalization processes in the extractive industry (figure 1).The largest mining companies are developing international projects and diversifying the regional risks of the mining business.This necessitated the development of integrated assessment systems that create unified and understandable tools for assessing mineral reserves and resources, regardless of the location of the assessment object itself.
This study analyzes and compares the classification features of various systems: Ukrainian classification [1], UNFC [2], The Petroleum Resources Management System (PRMS) [5], The Committee for Mineral Reserves International Reporting Standards (CRIRSCO) [6] in order to establish features that allow determining the social and environmental risks of subsoil use projects.
In the mining projects valuation, environmental and social risks, as well as ESG in general, are often used in the context of investment attractiveness, although stakeholders include not only the investment environment, but also customers, suppliers, company personnel, local populations, and governments, who are increasingly interested in safe (in all senses), and stable development of organizations and companies on their territory.The presence of these direct features in the classification systems indicates the orientation of the assessment standards to the comprehensive assessment of projects as such meeting the goals of sustainable development.
Below are comparisons of the basic parameters of classification systems (table 2): CRIRSCO (by International reporting template for the public reporting of exploration targets, exploration results, mineral resources, and mineral reserves [6], PRMS Petroleum resources management system update 2018 [5], The Norwegian Petroleum Directorate's resource classification system (NPD) 2016 [7] Ukrainian Classifications of reserves and resources for minerals of the State Subsoil Fund (SSF) [1].The features of classifications are given in the order in which they are considered in the system.
From the given comparison, it is fixed that the social and ecological viability of subsurface and natural resources projects is used as a direct feature only in the UNFC.In 1997, the UNFC was developed for solid minerals and conventional energy resources with the aim of becoming an ideal tool for comparing different regional and national standards.Currently, this classification has been expanded to include other types of resources.Today, the UNFC covers energy resources, including oil and gas; renewable energy projects; atomic energy resources; solid minerals; CO 2 storage projects; underground water; and anthropogenic resources, such as secondary resources recycled from production residues and wastes.An important feature is the systematization of information not only for geological and mining enterprises but also for all interested parties.Such parties are defined as international and regional organizations in the field of international research of mineral resources and energy (to facilitate the formulation of a consistent and visionary strategy of actions), national and local governments (for the sustainable management of national resources), companies and industrial enterprises (for the development of production and technologies, project management and financing), financial organizations and funds (for project management and financing) [2, 3].Thus, the UNFC itself is a tool for the effective management of national resources necessary for the realization of sustainable development goals, which enables all interested parties to find a common understanding of the development of resources and territories, as well as to compare projects in different types of resources and subsoil use.The development of the basic characteristics of the classification of reserves and resources in the UNFC from 1997 to 2020 is shown in the following table 3.
Geological knowledge
Other standards and classification systems have evolved in accordance with the more practical and rational objectives of providing reliable valuations for investors of all levels.The CRIRSCO standard notes that the extractive industry today is a global international business whose financial and operational success depends on the trust and confidence of investors [6].Unlike many other industries, geology and mining deal with mineral resources that are exhaustible and non-renewable.The main emphasis on the risks of mining projects in these standards is made on geological risks, which depend on the reliability of information about objects from the beginning of the geological study to the end of mining.Subsoil users are encouraged to effectively and transparently assess the risks associated with investments to ensure the high level of confidence necessary to sustain their activities [6].ESG criteria are taken into account as part of modifying factors that determine the possibility of converting mineral resources into reserves that are designed and have individual technical, technological, and organizational solutions for implementation, which forms their economic and environmental assessment in the relevant legal and regulatory conditions.In the hydrocarbon resource assessment standards 2018, the social and environmental component is also indirectly taken into account in the project success criteria ("chance" for commercial implementation) and uncertainty parameters.
Conclusions
The paper presents a comparison of classification features in the exploration and evaluation of mineral resources in domestic and international practice.This study analyzes and compares the classification features of various systems: The Ukrainian Classifications of reserves and resources for minerals of the State Subsoil Fund, The United Nations Framework Classification for Resources, The Petroleum Resources Management System, The Norwegian Petroleum Directorate's resource classification system, the Classification of the Committee for Mineral Reserves International Reporting Standards.
As a result, the most common feature of classifications is the level of geological study.This feature makes it possible to distinguish 4 groups of geological knowledge.Features of economic viability and status of the project, as a rule, are used in international practice.Important criteria, which are now reflected only in the UNFC, are the social and environmental viability of projects.This determines the importance of accounting for ESG in our valuation and resource accounting systems.
In recent years, social and environmental risks have become basic for the implementation of mining projects.Their growth can lead to the postponement of projects in time or the complete cancellation of decisions made because they did not meet social or environmental expectations, even if they met the requirements of quantity, quality, and profitability.Such changes must be reflected in classification systems for reserves and resources assessment that are used by all stakeholders to make management and investment decisions.Today, the social and ecological viability of subsurface and environmental projects is used as a direct feature only in the UNFC, and its inclusion took place in 2009 in the part of social factors, and in 2019 -in the part of environmental factors.This shows that the UNFC is a tool for the effective management of national resources, necessary for the realization of the goals of sustainable development, which enables all interested parties to find a common understanding of the development of resources and territories, as well as to compare projects in different types of resources and subsoil use.
In other international standards, as well as in the domestic classification of the State Subsoil Fund, the ESG criterion is taken into account indirectly, which is one of the key areas of development of these mineral resource evaluation systems.Such development can take place in two directions: 1) inclusion of social and ecological criteria of the direction as basic features, while this is possible both in the form of complex parameters and individual classes/subclasses; 2) use and improvement of existing methods of resource valuation, taking into account the environmental and social risks of the implementation of mining projects.
Figure 1 .
Figure 1.Development of international and national standards for mineral assessment in the period 1990-2020.
Table 2 .
Comparison of the basic features of the mineral resources classifications.
Table 3 .
Development of Principal elements of the UNFC. | 2023-11-05T16:12:25.355Z | 2023-10-01T00:00:00.000 | {
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2579442 | pes2o/s2orc | v3-fos-license | A dendritic nano-sized hexanuclear ruthenium(II) complex as a one- and two-photon luminescent tracking non-viral gene vector
Fluorescent tracking gene delivery could provide us with a better understanding of the critical steps in the transfection process. However, for in vivo tracking applications, a small diameter (<10 nm) is one of the rigorous requirements for tracking vectors. Herein, we have demonstrated a new paradigm for two-photon tracking gene delivery based on a dendritic nano-sized hexanuclear ruthenium(II) polypyridyl complex. Because this metallodendrimer has a multivalent periphery, the complex, which is 6.1 nm, showed high stability and excellent dispersibility and could stepwise condense DNA in vitro. With the outstanding photochemical properties of Ru(II) polypyridyl, this complex could track gene delivery in vivo using one- and two-photon imaging.
Herein, a dendritic hexanuclear Ru(II) polypyridyl complex based on polybenzimidazole ligands, Ru 6 L ( Fig. 1), was synthesized and developed as a one-and two-photon luminescent tracking non-viral gene vector. The photophysical properties and the size of Ru 6 L were characterized. After studying the interactions of Ru 6 L with DNA and the DNA condensation mechanism in vitro, the cellular uptake of both the complex and the Ru 6 L-DNA particles was investigated. Then, the complex was used as a one-and two-photon luminescent tracking non-viral gene vector. Finally, the transfection efficiency and cytotoxicity of the DNA condensates were evaluated by luciferase assays and MTT assays.
The photophysical data of Ru 6 L are summarized in Table S1. The absorption band at approximately 400-550 nm was assigned to metal-ligand charge transfer (MLCT) absorption ( Figure S4). The maximum emission wavelength (λ em ) was 612 nm, with a lifetime of 110 ns. The emission quantum yield was 0.027, and the largest two-photon absorption (TPA) cross-section was 175 GM at 830 nm with reference to rhodamine B ( Figure S5). The complex was a perfectly monodisperse nanoparticle with 12 positive charges derived from Ru 2+ ions ( Figure S6). The diameter of Ru 6 L was determined to be 6.1 nm by TEM (Fig. 2), which was similar to the hydrodynamic diameter 6.7 ± 0.3 nm determined by DLS ( Figure S6).
We investigated the complex's ability to condense DNA. The first study examined the migration of Ru 6 L-pBR 322 DNA by gel retardation assays with a load of 45 μ M (bp) DNA in each well. As shown in Fig. 3, with increasing the concentrations of Ru 6 L (0-6 μ M), pBR 322 DNA gradually diminished and was retained in the well at a + /− ratio (total positive charge/total negative charge) of 1.1. The primary DNA condensation-driving forces were considered electrostatic interactions by the multivalent periphery of Ru 6 L. The DNA binding ability was determined by a DNA titration approach with CT-DNA (calf thymus DNA). The binding constant was calculated to be 1.75 × 10 5 M −1 ( Figure S7).
The average hydrodynamic diameter of Ru 6 L-pBR 322 DNA particles at various + /− ratios in aqueous solution is shown in Fig. 3. The particle diameter was stable at approximately 140 nm when the + /− ratio was 4 or higher, while the zeta potential increased as the + /− ratio increased. To visually investigate the condensation process, we performed AFM of the Ru 6 L-2kb DNA particles at various + /− ratios (Fig. 4). When the + /− ratio was 1, local DNA bending, long-range cross-links and micro-loops were observed. Upon increasing the + /− ratio to 2, small particles were observed at the middle or the tail of the DNA duplex. As the + /− ratio increased, highly condensed DNA molecules were observed, and they condensed into compact hemispherical structures at a + /− ratio of 20. Ru 6 L-pBR 322 DNA particles at a + /− ratio of 20 were also observed as well-distributed DNA particles with a diameter from 50 to 90 nm ( Figure S8). In addition, we performed a DNA nuclease-catalyzed biodegradation assay ( Figure S9a), a photocleavage assay ( Figure S9b) and a continuous irradiation assay ( Figure S10) with Ru 6 L. The results showed that Ru 6 L displayed no photocleavage or photoreactivity toward DNA; in contrast, it protected DNA against nuclease-catalyzed biodegradation.
Before performing cellular uptake studies of Ru 6 L-DNA particles, we investigated the mechanism of cellular uptake of Ru 6 L using confocal microscopy ( Figure S11). Generally speaking, the cellular uptake of small molecules can occur through energy-independent (facilitated diffusion, passive diffusion) and energy-dependent (endocytosis, active transport) pathways. To determine whether Ru 6 L entered the cell via an energy-independent or energy-dependent transport pathway, HeLa cells were either incubated with Ru 6 L at 4 °C or pretreated with the metabolic inhibitors 2-deoxy-D-glucose and oligomycin. Figure S11b and S11c show that cellular luminescence was significantly suppressed in cases when the cells were incubated with the complex at 4 °C or pretreated with the metabolic inhibitors, indicating that Ru 6 L uptake followed an energy-dependent pathway. Endocytosis is well known as the most common energy-dependent pathway by which eukaryotic cells uptake extracellular materials. Endocytosis is also affected by temperature or adenosine triphosphate (ATP). We used the endocytic inhibitors chloroquine and NH 4 Cl to examine the role of this pathway in Ru 6 L uptake. As observed from the relative intensities and location of Ru 6 L in the cells after treatment with these inhibitors ( Figure S11d and S11e), they had no effect on the cellular uptake of Ru 6 L, which indicates that Ru 6 L is not taken up by living cells via an endocytosis pathway. This finding is not surprising because cellular uptake mechanisms are generally complex and diverse.
Flow cytometry was used to quantify the intracellular uptake of the DNA particles at the + /− ratio of 20 ( Figure S12). The result indicated that most of cells took up the DNA particles after a 4 h incubation in serum-free DMEM. We examined the localization of DNA particles in HeLa cells by TEM. TEM micrographs confirmed the uptake of the particles by endocytosis and showed that the DNA particles made HeLa cell form an endosome after they entered the HeLa cells (Fig. 5). Particles were also found in the cytoplasm, meaning that the particles successfully escaped from endosomes, which was important for transfection. The particle size was approximately 50 nm, as measured by AFM. The mechanism of cellular uptake of the DNA particles was also studied ( Figure S13). The cellular luminescence of Figure S13b which incubated with the particles at 4 °C was significantly suppressed, it means the particles entered the cell via an energy-independent transport pathway. After treatment with the metabolic inhibitors ( Figure S13c) and the endocytic inhibitors ( Figure S13d and S13e), no effect was found in the cells which treated with the metabolic inhibitors, but the cellular luminescence which treated with the endocytic inhibitors was suppressed, the results indicated that Ru 6 L-pEGFP DNA particles were taken up by living cells via an endocytosis pathway.
To investigate the intracellular behaviors of Ru 6 L-pEGFP DNA particles, one-and two-photon fluorescence microscopy was used to monitor the time-dependent transport and transfection of pEGFP DNA plasmids that were condensed by Ru 6 L (Fig. 6). We stained the nuclei of HeLa cells with Hoechst 33258; then, the DNA particles were added to the cells. In 20 min, the Ru 6 L-DNA particles attached to the cell membrane quickly due to their high positive zeta potential. After 4 h, particles were found in the cytoplasm. Then, the culture medium was replaced with fresh DMEM containing 10% fetal bovine serum (FBS). After another 12 h, EGFP expression was detected, and higher EGFP expression was found after 24 h. Similar behaviors were observed under one-photon and two-photon excitation. In addition, due to the two-photon absorption, the background signal was strongly suppressed, and a higher resolution image was obtained using two-photon confocal laser scanning microscopy.
The most commonly used method for intracellular plasmid trafficking is the fluorescent labeling of non-viral vectors with organic dyes 14,15 . It should be pointed out that to track the dynamic changes during a specific period of time, the dye must possess improved photostability and must be photostable under continual irradiation with light from fluorescent microscopes. However, most organic dyes have notable shortcomings, including poor water solubility, high toxicity to living cells and poor photostability. Organic dyes may also cause extensive cellular damage and unwanted background signal due the ultraviolet (UV) radiation required for their excitation and small Stokes shifts 26,27 . The short excitation wavelengths (< 650 nm) also inhibit the use of these materials in thick tissues or live animals due to the resultant short penetration depth 47 . In addition, the introduction of dyes may alter the delivery mechanisms and have increased side-effects 29 . The use of Ru 6 L with intrinsic two-photon luminescence as DNA carriers is an attractive solution to these problems because this complex exhibits near infrared (NIR) excitation wavelengths 48 .
We determined the relative transfection efficiency of this nonviral system by luciferase assays, and plasmid pGL3 was used a control vector (Fig. 7). As a control, applying DNA only resulted in a low level of luciferase expression. The luciferase expression was increased when Ru 6 L was used. The luciferase expression was stable when the + /− ratios of the particles ranged from 12 to 24, and the highest luciferase expression was in response to the + /− ratio of 20. Cytotoxicity of non-viral gene vectors is one of the major concerns of gene delivery. Therefore, we examined the viability of HeLa cells treated with the complex and Ru 6 L-pEGFP DNA particles (Fig. 8).
With increasing concentrations, the viability of the HeLa cells decreased slowly for both the complex and the particles. Although Ru 6 L exhibited the related cytotoxicity as the concentration increased, the viability of HeLa cells was more than 80% at a concentration of 5 μ M. The results indicated that this transfection system had relatively low cytotoxicity.
Conclusion
In the present study, we have designed and synthesized a dendritic nano-sized hexanuclear Ru(II) polypyridyl complex (Ru 6 L) based on polybenzimidazole ligands. This complex has a multivalent periphery and a nanosize of 6.1 nm. Gel retardation assay, TEM, AFM and dynamic light scattering studies show that Ru 6 L exhibits high stability and excellent dispersibility and could stepwise condense DNA in vitro. More interestingly, Ru 6 L was applied as a non-viral gene vector for tracking DNA delivery in live cells using one-and two-photon fluorescence microscopes. With two-photon microscopy, a high signal-to-noise contrast was achieved by irradiation with an 830 nm laser. Our work provides new insights into improving real-time tracking during gene delivery and transfection as well as important information for the design of multifunctional non-viral vectors.
Methods
All reagents were purchased from commercial sources and used without further purification unless otherwise specified. The plasmid pBR 322 DNA was obtained from MBI Fermentas, the plasmid pEGFP DNA was purchased from Clontech, and the plasmid pGL3 control vector and luciferase kid were obtained from Promega. Unless otherwise stated, the DNA concentrations are expressed in base pairs. All samples were prepared using distilled water that had been passed through a Millipore-Q ultra-purification system. The compounds 1,10-phenanthroline-5,6-dione 49 and [Ru(bpy) 2 ]Cl 2 .2H 2 O 50 were prepared according to literature methods. The complex was dissolved in DMSO prior to the experiments. Then, the calculated quantities of the complex solutions were added to the appropriate medium to yield a final DMSO concentration of less than 1% (v/v).
Microanalyses (C, H and N) were performed with a vario EL cube elemental analyzer. Infrared spectra were obtained with a Nicolet 170SX-FTIR spectrophotometer and KBr discs. Electrospray ionization mass spectra (ESI-MS) were recorded on an LCQ system (Finnigan MAT, USA).Fast atom bombardment mass spectrometry (FAB-MS) was recorded using a VG ZAB-HS. 1 H NMR spectra were recorded on a Varian-500 spectrometer at 25 °C. All chemical shifts are given relative to tetramethylsilane (TMS). The UV-Vis spectra were recorded on a Perkin-Elmer Lambda 850 spectrophotometer. Emission spectra were recorded on a Perkin-Elmer LS 55 spectrophotometer at room temperature (25 °C). Time-resolved emission measurements were conducted on an FLS 920 combined fluorescence-lifetime and steady-state spectrometer. Quantum yields of luminescence at room temperature (25 °C) were calculated according to literature procedures using [Ru(bpy) 3 ] 2+ (ϕ = 0.028 in aerated aqueous solution) as the reference emitter 51 . All date were processed using the Origin 8 software package.
Atomic force microscopy (AFM) images were obtained in air at room temperature with an SPA400 atomic force microscope unit and an SPI3800N control station (Seiko Instruments) operated in the tapping mode. Probes were made of a single silicon crystal with a cantilever length of 129 mm and a spring constant of 33-62N/m (OMCLAC160TS-W2, Olympus). Dynamic light scattering and zeta potential experiments were performed using dynamic laser light scattering equipment (DLS, Brooken Haven BI-200SM). Transmission electron micrographs were obtained with a JEM100CX electron microscope.
Synthesis of complex {[Ru
Dynamic laser light scattering equipment was used to determine the average hydrodynamic diameter and the zeta potential of Ru 6 L at a concentration of 5 μ M in distilled water that had been passed through a Millipore-Q ultra-purification system. Typically, 5 runs were measured for the solution, and the average of all runs is reported.
Determination of two-photon absorption cross-sections. The two-photon absorption (TPA) spectra of complex were determined over a broad spectral region by a two-photon induced luminescence (TPL) method relative to Rhodamine B in methanol as the standard. The two-photon luminescence data were acquired using an Opolette TM 355II (pulse width ≤ 100 fs, 80 MHz repetition rate, tuning range 730-890 nm, Spectra Physics, Inc., USA). Two-photon luminescence measurements were performed in fluorometric quartz cuvettes. The experimental luminescence excitation and detection conditions were conducted with negligible reabsorption processes, which can affect TPA measurements. The quadratic dependence of the two-photon induced luminescence intensity on the excitation power was verified at an excitation wavelength of 830 nm. The two-photon absorption cross-section of the complex was calculated at each wavelength according to Equation (1) where I is the integrated luminescence intensity, C is the concentration, n is the refractive index, and ϕ is the quantum yield. The subscript '1' refers to the reference samples, and '2' for the experimental samples.
Preparation of DNA particles. The DNA particles were prepared by incubating the mixtures containing DNA and Ru 6 L at the given + /− ratios in 50 mM Tris-HCl (Tris = Tris(hydroxymethyl) aminomethane) solution (pH = 7.4) or in cell culture, followed by vortexing for 30 min to allow equilibration at room temperature. Dynamic light scattering and zeta potential assay. Dynamic laser light scattering equipment was used to determine the average hydrodynamic diameter and the zeta potential of Ru 6 L-pBR 322 DNA particles at various + /− ratios in 50 mM Tris-HCl solution (pH = 7.4). Typically, 5 runs were measured for each solution, with the average of all the runs reported.
Gel retardation assay.
AFM imaging. The 2kb DNA fragment was generated by PCR using pBR 322 plasmid DNA as a template 53 . The condensation process of Ru 6 L-2kb DNA and the morphologies Ru 6 L-pBR 322 DNA were examined with AFM. Samples were dropwise-added (10 μ L) onto a mica substrate, which was freshly cleaved by pulling off the top sheets with tape. One min later, the substrate was spin coated (1400 rpm, 30 s) and rinsed with 20 μ L of distilled water. AFM images were obtained in air at room temperature. The images were captured in a 256 × 256 pixels format and analyzed with the software accompanying the imaging module. Cellular uptake of Ru6L-DNA particles. The cells were trypsinized, counted, and adjusted to 1 × 10 4 cells mL −1 , and 1 mL of the cell solution was added to each plate. After 24 h, the cell culture medium was replaced with 800 μ L serum-free DMEM. Ru 6 L-pEGFP DNA particles at the + /− ratio of 20, containing 1 μ g pEGFP DNA in 200 μ L serum-free DMEM, was added to the cells, and the cells were incubated at 37 °C for 4 h.
For flow cytometry, the cells were washed with PBS three times, trypsinized and centrifuged in PBS. Cells were harvested, and single cell suspensions in 0.5 mL PBS were prepared and subjected to flow cytometric analysis. A flow cytometer (Coulter Co. USA) was used to measure the fluorescent intensity, with excitation at 488 nm.
For TEM imaging analysis, cell processing was performed in situ, without displacement from the culture dish. Cells were fixed in 0.1 M PBS containing 2.5% glutaraldehyde and 4% paraformaldehyde for 1 h, rinsed with distilled water, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol (30, 60, 70, 90 and 100%), and embedded in epoxy resin. The resin was polymerized at 60 °C for 48 h. Ultrathin sections (50-75 nm) obtained with an LKB ultramicrotome were stained with 2% aqueous uranyl acetate and 2% aqueous lead citrate.
The mechanism of cellular uptake of Ru6L-DNA particles. The cells were trypsinized, counted, and adjusted to 1 × 10 5 cells mL −1 and 1 mL was added to five 35-mm 2 Petri dishes (MatTek, USA) for laser confocal microscopy. After 24 h, the first dish was incubated with Ru 6 L-pEGFP DNA particles at the + /− ratio of 20 at 37 °C for 4 h. The second dish was incubated with Ru 6 L-pEGFP DNA particles at the + /− ratio of 20 at 4 °C for 4 h. The third dish was pretreated with 50 mM 2-deoxy-D-glucose and 5 μ M oligomycin in PBS for 1 h at 37 °C and then incubated with Ru 6 L-pEGFP DNA particles at the + /− ratio of 20 at 37 °C for 4 h. The fourth and the fifth dishes were pretreated with endocytic inhibitors NH 4 Cl (50 mM), and chloroquine (50 μ M) for 30 min respectively, and then incubated with Ru 6 L-pEGFP DNA particles at the + /− ratio of 20 at 37 °C for 4 h.
After being washed with fresh PBS (pH = 7.0) three times, the cells were imaged on a Zeiss LSM 710 NLO confocal microscope (63 × /NA 1.4 oil immersion objective). For two-photon images, the excitation wavelength of the laser was 830 nm for Ru 6 L and the emission spectra were integrated over 580-630 nm (single channel).
Scientific RepoRts | 5:10707 | DOi: 10.1038/srep10707 One-and two-photon luminescent imaging. The cells were trypsinized, counted, and adjusted to 1 × 10 5 cells mL −1 , and 1 mL of the cell suspension was added to a 35-mm 2 Petri dish (MatTek, USA) for laser confocal microscopy. After 24 h, the cell culture medium was replaced with 800 μ L serum-free DMEM. Ru 6 L-pEGFP DNA particles at the + /− ratio of 20, containing 1 μ g pEGFP DNA in 200 μ L serum-fee DMEM, was added to the cells, and the cells were incubated at 37 °C for 4 h. Then, the medium was replaced with fresh DMEM containing 10% FBS, and the cells were incubated for various timed (20 min and 4, 12, and 24 h).
After being washed with fresh PBS (pH = 7.0) three times, the cells were imaged on a Zeiss LSM 710 NLO confocal microscope (63 × /NA 1.4 oil immersion objective). The excitation wavelength of the laser was 488 nm, and the emission spectra were integrated over 580-630 nm (single channel). For two-photon images, the excitation wavelength of the laser was 830 nm for Ru 6 L.
Luciferase assay. HeLa cells were seeded onto a 96-well cell-culture plate at a cell density of 1 × 10 4 cells per well and then incubated for 24 h. Cells were washed with PBS three times and then cultured with serum-free DMEM. Ru 6 L-pGL3 DNA control vector particles with increasing concentrations of the tested complex containing 0.2 μ g plasmid were added to the cells, and the cells were incubated at 37 °C for 4 h. The medium was then replaced with fresh DMEM with 10% FBS, and the cells were incubated for an additional 24 h. Cells were washed with PBS, harvested and treated for 30 min at 4 °C with end-over-end rotation in lysis buffer (50 mM Tris-HCl, pH = 7.5, 150 mM NaCl, 2% Triton X-100, 2% NP40). The luciferase assay was performed according to the manufacturer's protocol (Promega). Relative light units (RLUs) were measured with Varioskan Flash (Thermo Scientific, USA) and a GloMax TM 96 microplate luminometer (Promega, USA). | 2018-04-03T00:00:38.064Z | 2015-07-17T00:00:00.000 | {
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234918343 | pes2o/s2orc | v3-fos-license | Assessment of the Quality of Groundwater for Drinking Purpose in Rajanpur Tehsil, Pakistan
In this study, the quality of groundwater used for drinking purposes at Rajanpur Tehsil Pakistan is investigated. Water contamination causes several health problems to human being and the severity of problems mainly depends upon the level of pollutants present in water. Physical, chemical, and biological parameters of groundwater for twelve locations are analyzed using standard procedures. The obtained results are compared with the standards set by WHO. Most of the chemical parameters are found within the WHO standards except Arsenic, Calcium, and Sulfate at various locations. The Arsenic is found beyond WHO standards at nine locations with an increased percentage of 400% at three locations namely village Mehre Wala, Sehnwal, Near Basti Bhayan, and is also found beyond the permissible limit, i.e., upto 150% at six locations Sikhaniwala, Kot Mithan, Basti Nazru, Basti Asni, Jalal Pur, and Qutab Pul. Only three locations show satisfactory results, namely village shikarpur, village Murghai and Basti Lakha. The percentage of calcium at five locations such as Murghai, Basti Nazru, Basti Asni, Basti Lakha, and Chak Jalal Pur (Pull Pathan) is found to be 44%, 12%, 17.33%, 76%, and 49.33%, which is much higher than the WHO standards. Meanwhile, the Sulphate is also found to be beyond WHO standards at four locations, i.e. 30.8% for Mehrewala, 37.6% for Murghai, 34.8% for Basti Lakha and 34% for Chak Jalal Pur (Pull Pathan). Biological contamination including total coliform and fecal coliform are found to be beyond the limit at Sehanwala, Rangpur, Shikarpur, and Basti Nazru locations. The higher percentage of Arsenic, calcium, and sulfate in water indicate that the groundwater is not suitable for drinking purpose. Sustainable water treatment is highly recommended for drinking water at the identified contaminated location.
Introduction
P ure, clean, and germ-free water is essential for human health. The availability of fresh clean water is declining rapidly due to population growth and human activities. Deteriorated quality of water is accountable for the death of around 5 million children in developing countries [1]. It is reported that 30% of diseases are identified due to worse water quality consumption in Pakistan including diarrhea in children which is a major water borne disease [2]. Treatment of drinking water is essential to eliminate various diseasecausing toxic substances, odor-producing agents, and to make the water suitable for drinking and other domestic applications. Human welfare in modern life is linked with the quality of water consumed in everyday life. According to different health organization's standards, groundwater in several parts of Pakistan does not meet the quality standards for human consumption [3]. The groundwater quality depends on chemical, physical and bacterial characteristics, and thus determines the usefulness of water supply for the community [4].
The higher level of water pollution in rivers and groundwater results in biodiversity disturbance, water-borne diseases and reduced agricultural production. Not only water pollution but also the mismanagement of resources has sturdy socio-economic consequences on health and food security [5]. According to the report of State Bank of Pakistan, environmental health-related risks impose major health problems. Polluted water due to the presence of various organic, toxic elements, nitrates and nitrites can cause various health problems such as malfunctions of the human body, chronic diseases, and cancer [6]. Developing countries like Pakistan are facing water pollution problems due to the increase in population, industrialization, improper solid waste management and affluent discharge in canals and other water sources. The fast urbanization, deforestation, and land degradation have resulted in a sharp decline in water quality. Many cities in developing countries are facing problems like higher content of nutrient and organic material in potable water due to untreated domestic and industrial waste into the resources.
The objective of the current study is to assess and analyze groundwater quality of twelve wells at different depths and locations of district Rajanpur used for drinking purposes.
Material & Method
The study was carried out at Rajanpur Tehsil which is an administrative subdivision of Rajanpur district. The capital of the Tehsil is Rajanpur City. The Tehsil is administratively subdivided into 16 Union Councils, having a gross area of 2078 sq. km. It is situated on river Indus's right bank on Indus Highway. The coordinates of the Tehsil's Capital are 29°-05'-56" N and 70°-19'-25" E. Its distance from Dera Ghazi Khan is 112.5 km. The climate of Rajanpur is dry with low rainfall in the whole district. Water samples collected from twelve sites identified by the GPS coordinates are shown in Figure 1.
The samples of groundwater were collected from twelve different locations of eleven union councils, Tehsil Rajanpur and district Rajanpur. The samples were collected from the wells at different depths, mostly near the canals or expected sweet groundwater zones. The sterilized plastic bottles were washed with distilled water twice before the collection of the samples according to the standard methods [7] [8]. The water quality parameters were analyzed according to the procedures and the standard methods are summarized in Table 1. The samples collected from the selected locations of Rajanpur, as shown in Table 2, were analyzed in the environmental laboratory of Energy & Environment Engineering department QUEST Nawabshah.
Results & Discussion
The physical, chemical and biological parameters of water samples collected from the selected sites were determined in the laboratory to evaluate the suitability of water for drinking purpose by using analysis/equipment as mentioned in Table 1.
Physical Parameters
The physical parameters of groundwater were examined for twenty selected locations. Decaying of organic (vegetation) and inorganic (soil, stones, and rocks) matters impart color to water. Iron becomes yellow or red sediments. All analyzed samples were colorless. Taste and odors were also non-objectionable for all samples.
The presence of suspended particles in water is measured by turbidity level. The turbidity in water is the indication of cloudiness of water and is a key parameter for determining water quality [9]. The obtained results from turbidity analysis, as shown in Figure 2, were found within the permissible limit as recommended by WHO (i.e., 5 NTU) at all locations. Electrical conductivity (EC) is the ionic concentration present in water. Ions in solution cause the flow of electrical current. The results for EC were found between 400 to 1273 µS/cm (Figure 3) with an average value of 892 µS/cm. The results were within the acceptable limit of WHO.
Chemical Parameters
Various chemical parameters of this study are presented in Table 3. The analysis for all parameters was performed using standard methods The procedures are mentioned in Table 1.
The pH determines the acidic or alkaline nature of water [10]. The results of pH found for all locations were in the range 7.42-7.78 ( Figure 4) which are within the allowable limit of WHO standard, i.e., 6.5-8.5. Total Dissolved Solids (TDS) level of 300 mg/l and less is considered as the best level, the range 300-600 mg/l is considered as good, 600-1000 mg/l is considered as fair, and above 1000 mg/l is higher than the acceptable limit [11]. According to WHO standard, the higher boundary of TDS of potable water is 1000 mg/l. The TDS level was found within the permissible limits at all locations (shown in Figure 5). Chlorides specify the level of salinity in water. Chlorides in groundwater arises from several sources such as soil weathering, formation of saline lipids and precipitation of salt spray, salt for road melting, and contributions of wastewater. Chloride is not considered as harmful in drinking water, but its concerns are associated with the common relationship with a higher level of sodium. The obtained results for chloride were found within the allowable limit of WHO and NEQS standards, as shown in Figure 6.
The presence of Fluoride in groundwater is due to the weathering and leaching of fluoride bearing minerals from sediments and rocks found in foods such as tea, fish and rice, etc. The WHO recommended the value of fluoride for drinking water as 1.5 mg/l [12]. The results of fluoride were found between 0.15 mg/l to 0.4 mg/l with an average value of 0.31 mg/l ( Figure 7). The results of all locations for fluoride were found satisfactory.
Sulfates in water occurs naturally or from municipal and industrial discharges. A high-level of sulfate in drinking water may result in several health disorders and cause a problem in eye, skin, and scalp irritation, diarrhea, and dehydration. WHO's suggested safe limit for sulfate is 250 mg/l. The results of sulfate concentration, shown in Figure 8, range between 32 mg/l-344 mg/l at different locations. The sulfate level exceeded beyond the acceptable limit at four locations, i.e., at S2, S7, S10 and S11, indicating the potential health threat to human. The hardness test is performed for determining the properties of highly mineralized water. Hardness of water is the sum of calcium ions and magnesium ions, calculated by the analytical method. WHO's suggested hardness upper limit is 500 mg/l in drinking water. The results of hardness found range from 160 mg/l to 480 mg/l ( Figure 9). The results of all twelve locations were found within the safe limit as recommended by WHO and NEQS.
Calcium is an essential mineral for bones, cell physiology, and teeth. Surplus calcium intake is very harmful to human health like heart diseases, high blood pressure, irritated skin, diabetes, and reproductive failure [13] [14]. The results of this study showed that most of the calcium samples were within the WHO permissible limit of 75 mg/l. However, the analysis found higher calcium amounts than the acceptable limit at these locations, i.e., S7, S8, S9, S10 and S11, as shown in Figure 10.
Magnesium is one of the essential minerals in water for human health. It describes the quality of water for several applications [15]. The safe limit of Mg suggested by WHO is 150 mg/l for drinking water. Amount of Mg was found to be within the permissible limit at all locations ( Figure 11). A high level of Na changes the taste of water and can pose potential hazards to human health. Its safe limit suggested by WHO is 200 mg/l [11]. The results for Na concentration obtained at various locations for all twelve samples were found within the permissible limit ( Figure 12). The sodium concentration found at different locations were between 21-183 mg/l. Potassium (K) is an alkali element and is very helpful for hydration. The European Commission suggests potassium allowable limit for drinking water to be 12 mg/l [13]. Results of potassium were found within the safe limit ranging from 4.6 mg/l -9.8 mg/l with an average value of 6.5 mg/l, as shown in Figure 13. The consumption of water with alkalinity higher than permissible level may cause different health issues. The results of alkalinity were found between 3.1-7.1 m.mol/1, as shown in Figure 14, with an average value of 4.9 m.mol/1. Nitrogen is a natural source of nitrate in groundwater. Sources of nitrate are agricultural chemicals, sewage, and industrial effluents. Diseases associated with drinking water having higher nitrate levels are diabetes, thyroid disease, and gastric cancer. WHO recommends the nitrate maximum limit as 10 mg/l for drinking water. The nitrate results were found (Figure 15). Arsenic is known as a very toxic and cancer-causing chemical pollutant originate in drinking water. Longterm use of water contaminated with arsenic may cause skin cancer, lungs, bladder, kidney, pigmentation, weakness, numbness, muscular cramping, vomiting, and diarrhea [15]. The permissible limit of arsenic in drinking water suggested by WHO is 10 g/l. The results obtained for Arsenic range from 10 microgram/liter to 50 microgram/liter. The sample locations S2, S3, S4 were very high and locations S1, S6, S8, S9, S11, S12 were also found beyond the permissible limit. Only three locations S5, S7, S10 showed satisfactory results ( Figure 16).
According to the standards suggested by WHO, the concentrations of iron might be less than 0.3 mg/liter in drinking water. Iron concentration may increase due to iron salts used as agents for coagulant. The iron level found in all samples were within the permissible limit as recommended by WHO (Figure 17).
Biological Parameters
Biological parameters are considered as helpful indicators for water quality. Animal and human feces and agricultural activities are the major causes of microbial contamination of water [16]. According to WHO guidelines, potable water should be free from biological contamination, i.e., total coliform and fecal coliform 0/100 ml [11]. The presence of total coliform in water intake causes waterborne diseases such as gastrointestinal upset, flu, fever, abdominal cramps, and diarrhea. Agricultural runoff, effluent from a septic system or sewerage discharge, rainfall, and infiltration of domestic are the main sources of coliform in groundwater [17]. Results showed that the total coliform level exceeded the acceptable limit for samples, i.e., S3, S4, S5 S8 ( Figure 18). The results obtained for fecal coliforms were found higher than the acceptable limit
Conclusion
The present study showed the assessment of various water quality parameters. The observed results revealed that the quality of underground water that is being used by the people of Rajanpur Tehsil was satisfactory at some locations while contaminated at other studied locations. The arsenic, calcium, sulfate, total coliform, and fecal coliform were found beyond the WHO and NEQs standards at twelve locations. The Arsenic limit exceeded at nine locations: S1, S2, S3, S4, S6, S8, S9, S11 and S12. It exposed an alarming situation for those areas. Calcium was above the limit at locations: S7, S8, S9, S10 S11. Also, sulfate was found higher at locations: S2, S7, S10 S11. Total coliform and fecal coliform were found higher at locations: S3, S4, S5 S8. The use of fertilizer and pesticides such as herbicides, insecticides, fungicides, rodenticides, algicides in the agricultural land are the main sources for contamination groundwater. The higher level of calcium and sulfate originated due to the presence of gypsum in the ground. It was observed during the water sample collection that the improper disposal of municipal solid waste was also one of the reasons for groundwater contamination in rural areas of Tehsil Rajanpur. The effluent from the septic system, sewage discharge, and wild animal fecal underground penetration are the sources of biological contamination. Due to the consumption of contaminated water, the people of Tehsil Rajanpur were facing various health problems. Water treatment is highly recommended before the use of underground water for drinking purpose at the identified contaminated locations. | 2021-05-22T00:03:09.782Z | 2020-12-31T00:00:00.000 | {
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15721975 | pes2o/s2orc | v3-fos-license | About Fokker-Planck equation with measurable coefficients and applications to the fast diffusion equation
The object of this paper is the uniqueness for a $d$-dimensional Fokker-Planck type equation with non-homogeneous (possibly degenerated) measurable not necessarily bounded coefficients. We provide an application to the probabilistic representation of the so called Barenblatt solution of the fast diffusion equation which is the partial differential equation $\partial_t u = \partial^2_{xx} u^m$ with $m\in(0,1)$. Together with the mentioned Fokker-Planck equation, we make use of small time density estimates uniformly with respect to the initial condition
Introduction
The present paper is divided into three parts.i) A uniqueness result on a Fokker-Planck type equation with measurable non-negative (possibly degenerated) multidimensional unbounded coefficients.
ii) An application to the probabilistic representation of a fast diffusion equation.
iii) Some small time density estimates uniformly with respect to the initial condition.
In the whole paper T > 0 will stand for a fixed final time.In a one dimension space, the Fokker-Planck equation is of the type where a, b : [0, T ] × R → R are measurable locally bounded coefficients and µ is a finite real Borel measure.The Fokker-Planck equation for measures is a widely studied subject in the literature whether in finite or infinite dimension.Recent work in the case of time-dependent coefficients with some minimal regularity was done by [9,16,30] in the case d ≥ 1.In infinite dimension some interesting work was produced by [8].
In this paper we concentrate on the case of measurable (possibly) degenerate coefficients.Our interest is devoted to the irregularity of the diffusion coefficient, so we will set b = 0.A first result in that direction was produced in [7] where a was bounded, possibly degenerated, and the difference of two solutions was supposed to be in L 2 ([κ, T ] × R), for every κ > 0 (ASSUMPTION (A)).This result was applied to study the probabilistic representation of a porous media type equation with irregular coefficients.We will later come back to this point.We remark that it is not possible to obtain uniqueness without ASSUMPTION (A).In particular [7,Remark 3.11] provides two measure-valued solutions when a is time-homogeneous, continuous, with 1 a integrable in a neighborhood of zero.
One natural question is about what happens when a is not bounded and x ∈ R d .
A partial answer to this question is given in Theorem 3.1 which is probably the most important result of the paper; it is a generalization of [7,Theorem 3.8] where the inhomogeneous function a was bounded.Theorem 3.1 handles the multidimensional case and it allows a to be unbounded.
An application of Theorem 3.1 concerns the parabolic problem: where δ 0 is the Dirac measure at zero and u m denotes u|u| m−1 .It is well known that, for m > 1, there exists an exact solution to (1.2), the so-called Barenblatt's density, see [3].Its explicit formula is recalled for instance in [34,Chapter 4] and more precisely in [4,Section 6.1].Equation (1.2) is the classical porous medium equation.
In this paper, we focus on (1.2) when m ∈]0, 1[: the fast diffusion equation.In fact, an analogous Barenblatt type solution also exists in this case, see [34,Chapter 4] and references therein; it is given by the expression where 1−m dx. (1.4) 2) is a particular case of the so-called generalized porous media type equation xx β (u(t, x)) , t ∈]0, T ], u(0, x) = u 0 (dx), x ∈ R, (1.5) where β : R → R is a monotone non-decreasing function such that β(0) = 0 and u 0 is a finite measure.When β(u) = u m , m ∈]0, 1[ and u 0 = δ 0 , two difficulties arise: first, the coefficient β is of singular type since it is not locally Lipschitz, second, the initial condition is a measure.Another type of singular coefficient is β(u) = H(u − u c )u, where H is a Heaviside function and u c > 0 is some critical value, see e.g.[2].Problem (1.2) with m ∈]0, 1[ was studied by several authors.For a bounded integrable function as initial condition, the equation in (1.2) is well-stated in the sense of distributions, as a by product of the classical papers [10,6] on (1.5) with general monotonous coefficient β.
Electron.J. Probab.0 (2012), no.0, 1-28.ejp.ejpecp.org When the initial data is locally integrable, existence was proved by [19].[11] extended the validity of this result when u 0 is a finite Radon measure in a bounded domain, [29] established existence when u 0 is a locally finite measure in the whole space.The Barenblatt's solution is an extended continuous solution as defined in [13,14]; [14,Theorem 5.2] showed uniqueness in that class.[23,Theorem 3.6] showed existence in a bounded domain of solutions to the fast diffusion equation perturbed by a right-hand side source term, being a general finite and positive Borel measure.As far as we know, there is no uniqueness argument in the literature whenever the initial condition is a finite measure in the general sense of distributions.Among recent contributions, [15] investigated the large time behavior of solutions to (1.2).
The present paper provides the probabilistic representation of the (Barenblatt's) solution of (1.2) and exploits this fact in order to approach it via a Monte Carlo simulation with an L 2 error around 10 −3 .We make use of the probabilistic procedure developed in [4, Section 4] and we compare it to the exact form of the solution U of (1.2) which is given by the explicit formulae (1.3)- (1.4).The target of [4] was the case β(u) = H(u − u c )u; in that paper those techniques were compared with a deterministic numerical analysis recently developed in [12] which was very performing in that target case.At this stage, the implementation of the same deterministic method for the fast diffusion equation does not give satisfying results; this constitutes a further justification for the probabilistic representation.
We define where W is a Brownian motion on some suitable filtered probability space (Ω, F, (F t ) t≥0 , P ).
To the best of our knowledge, the first author who considered a probabilistic representation of a solution of (1.5) was H. P. Jr. McKean ([26]), particularly in relation with the so-called propagation of chaos.In his case β was smooth, but the equation also included a first order coefficient.From then on, literature steadily grew and nowadays there is a vast amount of contributions to the subject, especially when the nonlinearity is in the first order part, as e.g. in Burgers' equation.We refer the reader to the excellent survey papers [33] and [18].A probabilistic interpretation of (1.5) when β(u) = u.|u|m−1 , m > 1, was provided for instance in [5].Recent developments related to chaos propagation when β(u) = u 2 and β(u) = u m , m > 1 were proposed in [28] and [17].The probabilistic representation in the case of possibly discontinuous β was treated in [7] when β is non-degenerate and in [2] when β is degenerate; the latter case includes the case β(u) = H(u − u c )u.
As a preamble to the probabilistic representation we make a simple, yet crucial observation.Let W be a standard Brownian motion.Proposition 1.1.Let β : R → R such that β(u) = Φ 2 (u).u,Φ : R → R + and u 0 be a probability real measure.
Let Y be a solution to the problem (1.7) Electron.J. Probab.0 (2012), no.0, 1-28.ejp.ejpecp.org Proof of the above result is based on the following lemma.
Consider the function t → ρ(t, •) from [0, T ] to the space of finite real measures M(R), defined as ρ(t, •) being the law of Y t .Then ρ is a solution, in the sense of distributions Proof of Lemma 1.2.This is a classical result, see for instance [32,Chapter 4].The proof is based on an application of Itô's formula to ϕ(Y t ), ϕ ∈ S(R).
Proof of Proposition 1.1.We set a(s, y) = Φ 2 (u(s, y)).We apply Lemma 1.2 setting ρ(t, dy) = u(t, y)dy, t ∈]0, T ], and ρ(0, •) = u 0 .When u 0 is the Dirac measure at zero and β(u) = u m , with m ∈] 3 5 , 1[, Theorem 5.7 states the converse of Proposition 1.1, providing a process Y which is the unique (weak) solution of (1.6).The first step consists in reducing the proof of that Theorem to the proof of Proposition 5.3 where the Dirac measure, as initial condition of (1.2), is replaced by the function U(κ, •), 0 < κ ≤ T .This corresponds to the shifted Barenblatt's solution along a time κ, which will be denoted by U. Also, in this case Proposition 5.3 provides an unique strong solution of the corresponding non-linear SDE.That reduction is possible through a weak convergence argument of the solutions given by Proposition 5.3 when κ → 0. The idea of the proof of Proposition 5.3 is the following.Let W be a standard Brownian motion and Y 0 be a r.v.distributed as U(κ, •) admits a unique strong solution.The marginal laws of (Y t ) and U can be shown to be both solutions to (1.8) for a(s, y) = (U(s, y)) m−1 ; that a will be denoted in the sequel by ā.The leading argument of the proof is carried by Theorem 3.1 which states uniqueness for measure valued solutions of the Fokker-Planck type PDE (1.8) under some Hypothesis(B).More precisely, to conclude that the marginal laws of (Y t ) and U coincide via Theorem 3.1, we show that they both verify the so-called Hypothesis(B2).In order to prove that for U, we will make use of Lemma 4.2.The verification of Hypothesis(B2) for the marginal laws of Y is more involved.It makes use of a small time (uniformly with respect to the initial condition) upper bound for the density of an inhomogeneous diffusion flow with linear growth (unbounded) smooth coefficients, even though the diffusion term is non-degenerate and all the derivatives are bounded.This is the object of Proposition 5.1, the proof of which is based on an application of Malliavin calculus.In our opinion this result alone is of interest as we were not able to find it in the literature.When the paper was practically finished we discovered an interesting recent result of M. Pierre, presented in [20,Chapter 6], obtained independently.This result holds in dimension 1 when the coefficients are locally bounded, non-degenerate and the initial condition has a first moment.In this case, the hypothesis of type (B) is not needed.
In particular it allows one to establish Proposition 5.3, but not Theorem 5.7 where the coefficients are not locally bounded on [0, T ] × R.
Electron.J. Probab.0 (2012), no.0, 1-28. ejp.ejpecp.org The paper is organized as follows.Section 2 is devoted to basic notations.Section 3 is concentrated on Theorem 3.1 which concerns uniqueness for the deterministic, time inhomogeneous, Fokker-Planck type equation.Section 4 presents some properties of the Barenblatt's solution U to (1.2).The probabilistic representation of U is treated in Section 5. Proposition 5.1 performs small time density estimates for timeinhomogeneous diffusions, the proof of which is located in the Appendix.Finally, Section 6 is devoted to numerical experiments.
Preliminaries
We start with some basic analytical framework.In the whole paper d will be a strictly positive integer.If f : R d → R is a bounded function we will denote f ∞ = sup By S(R d ) we denote the space of rapidly decreasing infinitely differentiable functions ϕ : R d → R, by S (R d ) its dual (the space of tempered distributions).We denote by M(R d ) the set of finite Borel measures on R d .If x ∈ R d , |x| will denote the usual Euclidean norm.
For ε > 0, let K ε be the Green's function of ε − ∆, that is the kernel of the operator In particular, for all ϕ ∈ L 2 (R d ), we have For more information about the corresponding analysis, the reader can consult [31].If ϕ ∈ C 2 (R d ) S (R d ), then (ε−∆)ϕ coincides with the classical associated PDE operator evaluated at ϕ. Definition 2.1.We will say that a function ψ : Definition 2.2.We will say that a function ψ : [0, T ] × R → R has linear growth (with respect to the second variable) if there is a constant with initial condition z(0, •) = z 0 if, for every t ∈ [0, T ] and φ ∈ S(R), we have (2.2)
Uniqueness for the Fokker-Planck equation
We now state the main result of the paper which concerns uniqueness for the Fokker-Planck type equation with measurable, time-dependent, (possibly degenerated and unbounded) coefficients.It generalizes [7,Theorem 3.8] where the coefficients were bounded and one-dimensional.
The theorem below holds with two classes of hypotheses: (B1), operating in the multidimensional case, and (B2), more specifically in the one-dimensional case.Theorem 3.1.Let a be a Borel nonnegative function on [0, T ] × R d .Let z i : [0, T ] → M(R d ), i = 1, 2, be continuous with respect to the weak topology on finite measures on M(R d ).Let z 0 be an element of M(R d ).Suppose that both z 1 and z 2 solve the problem ∂ t z = ∆(az) in the sense of distributions with initial condition z(0, •) = z 0 .
Then z := (z 1 −z 2 )(t, •) is identically zero for every t under the following requirement.
(B2) We suppose d = 1.For every t 0 > 0, we have 1.If a is bounded then the first item of Hypothesis(B1) implies the second one.
1.If a is non-degenerate, the third assumption of Hypothesis(B2) implies the first one.
Proof of Theorem 3.1.Let z 1 , z 2 be two solutions of (2.2); we set z := z 1 − z 2 .We evaluate, for every t ∈ [0, T ], the quantity Similarly to the first part of the proof of [7, Theorem 3.8], assuming we can show that we are able to prove that z(t) ≡ 0 for all t ∈]0, T ].We explain this fact.Electron.J. Probab.0 (2012), no.0, 1-28. ejp.ejpecp.org Since the two terms of the above sum are non-negative, if (3.1) holds, then •) → 0, in the sense of distributions, as ε goes to zero.Therefore z ≡ 0.
(iii) The Dirac measure δ 0 is the initial trace of U, in the sense that for every γ : R → R, continuous and bounded.
Proof of Proposition 4.1.(i) This is a well known fact which can be established by inspection.
(ii) For M ≥ 1, we consider a sequence of smooth functions (ϕ M ), such that Letting M → +∞, by Lebesgue's dominated convergence theorem, the left-hand side of (4.3) converges to R U(t, x)dx.The integral on the right-hand side of (4.3) is bounded by The last integral on the right is finite as m 1 − m > 0, for every m ∈]0, 1[.Therefore the integral in the right-hand side of (4.3) goes to zero as M → +∞.This concludes the proof of the second item of Proposition 4.1.
Note that the second item of Proposition 4.1 determines the explicit expression of the constant D. (i) Suppose that 1 3 < m < 1 .Then there is p ≥ 2 and a constant C p (depending on T ) such that for 0 (ii) In particular, taking p = 2 in (4.4), we get again when m belongs to ] 1 3 , 1[.
Proof of Lemma 4.2.
(ii) is a particular case of (i) and (iii) follows by similar arguments as for the proof of (i).
In particular we have ejp.ejpecp.org
The probabilistic representation of the fast diffusion equation
We are now interested in a non-linear stochastic differential equation rendering the probabilistic representation related to (1.2) and given by (1.6).Suppose for a moment that Y 0 is a random variable distributed according to δ 0 , so Y 0 = 0 a.s.We recall that, if there exists a process Y being a solution in law of (1.6), then Proposition 1.1 implies that u solves (1.2) in the sense of distributions.
In this subsection we shall prove existence and uniqueness of solutions in law for (1.6).In this respect we first state a tool, given by Proposition 5.1 below, concerning the existence of an upper bound for the marginal law densities of the solution Y of an inhomogeneous SDE with unbounded coefficients.This result has an independent interest. (5.1) Then, for every s > 0, the law of Y s admits a density denoted p s (x 0 , •).
Moreover, we have where K is a constant which depends on σ ∞ , b ∞ and T but not on x 0 .3. If σ and b have polynomial growth and are time-homogeneous, various estimates are given in [25].However the behavior is of type O(t Let Y κ be a random variable distributed according to u 0,κ .We are interested in the following result.Proposition 5.3.Assume that m ∈] 3 5 , 1[.Let B be a classical Brownian motion independent of Y κ .Then there exists a unique (strong In particular pathwise uniqueness holds.
Proof of Proposition 5.3.Let W be a classical Brownian motion on some filtered probability space.Given the function U, defined in (4.10), we construct below a unique process Y strong solution of (5.6) In fact, Φ(U) is continuous, smooth with respect to the space parameter and all the space derivatives of order greater or equal than one are bounded; in particular Φ(U) is Lipschitz and it has linear growth.Therefore (5.5) admits a strong solution.
To conclude it remains to prove that U(t, y)dy is the law of Y t , ∀t ∈ [0, T −κ]; in particular the law of the r.v.Y t admits a density.For this we will apply Theorem 3.1 for which we need to check the validity of Hypothesis(B2) when a = ā and for z := z 1 − z 2 , where z 1 := ρ and z 2 := U.By additivity this will be of course fulfilled if we prove it separately for z := ρ and z := U, which are both solutions to (5.7).
Lemma 5.5.Let ψ : [0, T ] × R → R + , continuous (not necessarily bounded) such that ψ(t, •) is smooth with bounded derivatives of orders greater or equal than one.We also suppose ψ to be non-degenerate.
If X 0 = x 0 , where x 0 is a real number, then Proposition 5.1 implies that, for every t ∈]0, T ], the law of X t admits a density p t (x 0 , •).Consequently, if the law of X 0 is u 0,κ (x)dx, for every t ∈]0, T ], the law of X t has a density given by ν(t, x) = R u 0,κ (x 0 )p t (x 0 , x)dx 0 . (5.9) Using (5.9) we get (5.10) the latter inequality is valid because of (4.7) in Lemma 4.2.In the sequel of the proof, the constants K 2 , K 3 , K 4 will only depend on t 0 , T and ψ.Furthermore Since ψ has linear growth, this expression is bounded by (5.11) (5.11) follows because of (5.10).Besides, by Burkholder-Davis-Gundy and Jensen's inequalities, taking into account the linear growth of ψ, it follows that Then, by Gronwall's lemma, there is another constant K 4 such that (5.12) Finally (5.11), (5.12) and (5.10) allow us to conclude the proof.
We are now ready to provide the probabilistic representation related to function U which in fact is only a solution in law of (1.6).
Definition 5.6.We say that (1.6) admits a weak (in law) solution if there is a probability space (Ω, F, P), a Brownian motion (W t ) t≥0 and a process (Y t ) t≥0 such that the system (1.6) holds.(1.6) admits uniqueness in law if, given (W 1 , Y 1 ), (W 2 , Y 2 ) solving (1.6) on some related probability space, it follows that Y 1 and Y 2 have the same law.Proof of Theorem 5.7.First we start with the existence of a weak solution for (1.6).
Let U be again the (Barenblatt's) solution of (1.2).We consider the solution (Y κ t ) t∈[κ,T ] provided by Corollary 5.4 extended to [0, κ], setting Y κ t = Y κ , t ∈ [0, κ].We prove that the laws of processes Y κ are tight.For this we implement the classical Kolmogorov's criterion, see [22,Section 2.4,Problem 4.11 ].We will show the existence of p > where C p will stand for a constant (not always the same), depending on p and T but not on κ.Let s, t ∈]0, T ].Let p > 2. By Burkholder-Davis-Gundy inequality we obtain Then, using Jensen's inequality and the fact that U(r, •) is the law density of Y κ r , r ≥ κ, Consequently there is a subsequence Y n := Y κn converging in law (as C([0, T ])−valued random elements) to some process Y .Let P n be the corresponding laws on the canonical space Ω = C([0, T ]) equipped with the Borel σ-field.Y will denote the canonical process Y t (ω) = ω(t).Let P be the weak limit of (P n ).
1) We first observe that the marginal laws of Y under P n converge to the marginal law of Y under P .Let t ∈]0, T ].If the sequence (κ n ) is lower than t, then the law of Y t under P n equals the constant law U(t, x)dx.Consequently, for every t ∈]0, T ], the law of Y t under P is U(t, x)dx.
2) We now prove that Y is a (weak) solution of (1.6), under P .By similar arguments as for the classical stochastic differential equations, see [32,Chapter 6], it is enough to prove that Y (under P ) fulfills the martingale problem i.e., for every f ∈ C 2 b (R), the process is an (F s )-martingale, where (F s ) is the canonical filtration associated with Y .C 2 b (R) stands for the set {f ∈ C 2 (R)|f, f , f bounded }.Let E (resp.E n ) be the expectation operator with respect to P (resp.P n ).Let s, t ∈ [0, T ] with s < t and R = R(Y r , r ≤ s) be an F s −measurable, bounded and continuous (on C([0, T ])) random variable.In order to show the martingale property (MP) of Y , we have to prove that (5.15) Electron.J. Probab.0 (2012), no.0, 1-28.
ejp.ejpecp.org
We first consider the case when s > 0. There is n ≥ n 0 , such that κ n < s.Let f ∈ C 2 b (R); since (Y s ) s≥κn , under P n , are still martingales we have (5. 16) We are able to prove that (5.15) follows from (5.16).Let ε > 0 and N > 0 such that U m (r, y)dy ≤ ε, (5.17 where C is the linear growth constant of Φ 2 • U in the sense of Definition 2.2.In order to conclude, passing to the limit in (5.16), we will only have to show that where F ( ) but not bounded.The left-hand side of (5.18) equals where Since κ n < s, for N large enough, we get taking into account (5.17) and the second item of Lemma 4.2.For fixed N , chosen in (5.17), we have lim n→+∞ E 2 (n, N ) = 0, since F N is bounded and continuous.Again, since the law density under P of Y t , t ≥ s , is U(t, •), similarly as for (5.20), we obtain since ε > 0 is arbitrary, (5.18) is established.So (5.15) is verified for s > 0.
3) Now, we consider the case when s = 0. We first prove that using Lebesgue's dominated convergence theorem and (5.21).Consequently we obtain (5.22) It remains to show that Y 0 = 0 a.s.This follows because Y t → Y 0 a.s., and also in law (to δ 0 ), by the third item of Proposition 4.1.Finally we have shown that the limiting process Y verifies (MP), which proves the existence of solutions to (1.6).4) We now prove uniqueness.Since U is fixed, only uniqueness for the first line of equation (1.6) has to be established.Let (Y i t ) t∈[0,T ] , i = 1, 2, be two solutions.In order to show that the laws of Y 1 and Y 2 are identical, according to [21, Lemma 2.5], we will verify that their finite marginal distributions are the same.For this we consider 0 = t 0 < t 1 < . . .< t N = T .Let 0 < κ < t 1 .Obviously we have Y i t 0 = 0 a.s., in the corresponding probability space, ∀i ∈ {1, 2}.
Numerical experiments
In order to avoid singularity problems due to the initial condition being a Dirac delta function, we will consider a time translation of U, denoted v, and defined by v still solves equation (1.2), for m ∈]0, 1[, but with now a smooth initial data given by Indeed, we have the following formula where α, k and D are still given by (1.4).
We now wish to compare the exact solution of problem (1.2) to a numerical probabilistic solution.In fact, in order to perform such approximated solutions, we use the algorithm described in Sections 4 of [4] (implemented in Matlab).We focus on the case We start with some notations for the Malliavin calculus.The set D ∞ represents the classical Sobolev-Malliavin space of smooth test random variables.D 1,2 is defined in the lines after [27, Lemma 1.2.2] and L 1,2 is introduced in [27, Definition 1.3.2].See also [24] for a complete monograph on Malliavin calculus.We state a preliminary result.Proposition 7.1.Let N be a non-negative random variable.Suppose, for every p ≥ 1, the existence of constants C(p) and 0 (p) such that x −p dF (x).
(7.1) implies that I 1 and I 2 are well-defined.Indeed, on one hand, applying integration by parts on I 1 , we get moreover, there is a constant C(p) such that On the other hand, again (7.1) says that Consequently, using (7.4) and (7.5) and coming back to (7.3), (7.2) is established.
Proof of Proposition 5.1.In this proof σ (resp.b ) stands for , be the solution of (5.1).According to [27, Theorem 2. [27,Theorem 2.3.1], the law of Y s admits a density that we denote by p s (x 0 , •).
The second step consists in a re-scaling, transforming the time s into a noise multiplicative parameter λ; ), where In particular, Y s ∼ Y λ 1 .By previous arguments, for every t > 0, Y λ t ∈ D ∞ and its law admits a density denoted by p λ t (x 0 , •).Our aim consists in showing the existence of a constant K such that where K is a constant which does not depend on x 0 and λ.In fact, we will prove that, for every λ ∈]0, We set In fact, we will have attained (7.7), if we show We express the equation fulfilled by Z; it yields where, for every (r, z) ∈ [0, 1] × R, we set σ λ (r, z) = σ(rλ 2 , λz + x 0 ), and b λ (r, z) = λb(rλ 2 , λz + x 0 ).
At this stage we state the following lemma.
1.For simplicity, in the whole proof of Proposition 5.1, we will set T = 1.
2. Since there is no more ambiguity, we will use again the letter s in the considered integrals.
Then, using Jensen's inequality, we get Then, coming back to (7.18) and using (7.23), we obtain where . Finally, using Proposition 7.1, the result follows.
We go on with the proof of Proposition 5.1 taking into account the considerations before Lemma 7.4.In fact [27, Proposition 2.1.1]allows us to express, for fixed t ∈]0, 1], using Cauchy-Schwarz inequality, it implies that (7.26) According to (1.48) in [27], (7.26) implies Now we state a result that estimates the two terms in the right-hand side of (7.27).Indeed, we have the following.
In fact, we have On the other hand, we get Therefore, coming back to (7.30) and using (7.31), we obtain that Electron.J. Probab.0 (2012), no.0, 1-28.ejp.ejpecp.orgwhere with D s1 G den given in (7.32).In the sequel we will enumerate constants K 1 to K 20 ; all those will not depend on x 0 or t, but eventually on T , σ and b.We start estimating J 1 .Since σ is bounded, by Cauchy-Schwarz inequality, we have .
Since σ has linear growth, Lemma 7.2 and a further use of Cauchy-Schwarz inequality imply that, J 1 is bounded by .
Therefore, by Proposition 7.5 and Lemma 7.4, we obtain that (7.34) We go on with the analysis of J 2 .Since σ , σ are bounded, we have Returning to the proof of Proposition 5.1 and substituting in (7.27) the right-hand side of the first and the second item of Proposition 7.6, the inequality (5.2) is verified.Finally this concludes the proof of Proposition 5.1.Electron.J. Probab.0 (2012), no.0, 1-28. ejp.ejpecp.org
Definition 2 . 3 .
Let a : [0, T ] × R d → R + be a Borel function, z 0 ∈ M(R d ).A (weakly measurable) function z : [0, T ] → M(R d ) is said to be a solution in the sense of distributions of ∂ t z = ∆(az)
Hypothesis(B2) is always verified by Remark 3.2; the first item of Hypothesis(B2) implies the third one.So Theorem 3.1 is a strict generalization of [7, Theorem 3.8].4. Let (z(t, •), t ∈ [0, T ]) be the marginal law densities of a stochastic process Y solving
Remark 5. 2 . 1 . 2 .
The proof of Proposition 5.1 above is given in Appendix 7.1.If σ and b is bounded, the classical Aronson's estimates implies that (5.2) holds even without the |x 0 | 4 multiplicative term.If σ and b are unbounded,[1] provides an adaptation of Aronson's estimates; unfortunately they first considered timehomogeneous coefficients, and also their result does not imply (5.2).
where C 1 and C 2 depend on T , σ ∞ and b ∞ , but not on x 0 .whereG den =< DZ t , DZ t > .Since σ has linear growth, by Lemma 7.2 and using again Cauchy-Schwarz inequality, there is a constant C 1 such that | 2012-09-18T06:13:24.000Z | 2011-11-28T00:00:00.000 | {
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195857046 | pes2o/s2orc | v3-fos-license | Type 2 Diabetes: Multiple Genes, Multiple Diseases
Purpose of Review Type 2 diabetes (T2D), which accounts for the vast majority of diabetes cases, is essentially a diagnosis of exclusion in current clinical practice. Therefore, it is not surprising that T2D is heterogenous in terms of patients’ clinical presentation, disease course, and response to treatment. This review summarizes published attempts to improve diabetes subclassification, with a particular focus on the role of genetics. Recent Findings A handful of diabetes subclassification schemas have been proposed using clinical data (patient characteristics and laboratory values), with some subgroups associated with distinct management trends or complication risks. However, phenotypically driven classifications suffer from dependencies on time of variable measurement and are not readily linked to disease mechanism. Germline genetic data, in contrast, are essentially unchanged over a person’s lifetime and rooted in mechanism. Clustering of T2D genetic loci has identified at least five groupings of loci representing mechanisms of disease that may aid in deconstructing heterogeneity of T2D, but further work is needed to determine clinical utility. Summary Exciting progress in subclassification of diabetes has demonstrated initial steps in deconstructing disease heterogeneity. Incorporation of genetics into classification schemas will require additional research but has the potential to improve our understanding and management of T2D, both as a single disease and as a part of an integrated metabolic disease network.
Introduction
In current clinical practice, when a patient develops elevated blood glucose indicative of diabetes, the diagnostic process of determining the diabetes "type" typically involves initially assessing for causes other than type 2 diabetes (T2D). For example, detection of autoantibodies may point to type 1 diabetes (T1D) or latent autoimmune diabetes in adults (LADA) or the presence of glucocortic o i d s o n t h e m e d i c a t i o n l i s t m i g h t s u g g e s t glucocorticoid-induced hyperglycemia. If a specific reason for hyperglycemia is not identified, a patient will generally then be considered to have T2D. Indeed, in practice, T2D is a diagnosis of exclusion, yet it currently is estimated to account for approximately 90% of all cases of diabetes [1]. It is not surprising, therefore, that T2D is a highly heterogenous condition with patients varying considerably in clinical presentation and response to treatment [2]. The heterogeneity observed among patients with T2D likely reflects variable contributions from diverse genetic and environmental factors [3], and ongoing efforts have aimed to utilize clinical and molecular data to develop a rational and reproducible categorization of diabetes. The goal of such subclassification is not only to refine patient diagnosis but also to better inform clinical management, specifically as it relates to prevention of diabetes complications. This review will summarize the diabetes-subtyping schemas that have been proposed as tools for deconstructing the heterogeneity of disease, with a particular focus on the role of genetics and its potential to shape our understanding and management of T2D, both as a single disease and as a part of an integrated metabolic disease network.
Evidence for Type 2 Diabetes Constituting "Multiple Diseases" The stereotypical phenotype of a patient with T2D is someone obese with evidence of insulin resistance; however, diabetologists know well that not all patients with T2D fit this mold. Likewise, not all patients with presumed T1D present with diabetic ketoacidosis (DKA) and have positive islet autoantibodies [1]. This recognition of disease heterogeneity has prompted several efforts to refine the classification of diabetes.
In 2003, Maldonado et al. presented the "AB" scheme, which was proposed to categorize patients presenting with DKA [4], and provided a useful construct for illustrating the diversity of these patients, who traditionally would have been assumed to have T1D. In 103 patients of various ethnic backgrounds who were admitted to the hospital with DKA, the authors assessed for presence of islet autoantibodies (A+/−) and evidence of beta-cell functional reserve (B+/−). They found that 50% of the patients were A−B+, 22% A−B−, 17% A+B−, and 11% A+B+ [4]. With only 17% of patients displaying antibody positivity and reduced beta-cell functional reserve (typical of T1D), the majority of these patients did not fit with the classic phenotypic picture associated with DKA. Furthermore, the substantial representation beyond both A−B+ (typical of T2D) and A+B− (typical of T1D) clearly demonstrated that patients with diabetes developing DKA did not fit neatly into well-established disease categories and that different pathophysiologies underlay their diabetes [5]. Of additional note, individuals in the B+ and B− groups also differed significantly by age of onset, glycemic control, and duration of insulin dependence, suggesting that recognition of subtype had clinical implications [4]. The utility in capturing beta-cell function in classifying diabetes "types" was also supported by a "beta-cell centric classification schema" later proposed by Schwartz et al., which conceptualized at least 11 pathways causing beta-cell dysfunction, each of which the authors envisioned could be targeted using a tailored treatment strategy [2].
Diversity among clinical phenotypes leading to development of diabetes was also evidenced in the work of Hulman et al. analyzing multi-point oral glucose tolerance tests (OGTT) in 5861 individuals without diabetes for whom longitudinal data was available. While typically only the fasting and two-hour time points of the OGTT are considered for diagnosing diabetes in non-pregnant adults, this analysis incorporated a third 30-min time point and fit latent class mixedeffects models across the three time points to identify four distinct glucose trajectory patterns [6]. Of particular interest was a subgroup (group 3) comprising 13% of individuals who had non-elevated 2-h glucose values, but elevated 30-min values; after up to 13 years of follow-up, individuals in group 3 were found to have a fourfold increased risk of developing diabetes and almost twofold all-cause mortality risk compared with individuals with similarly low 2-h glucose values, but who had non-elevated 30-min glucose readings (group 1). Compared with group 1, group 3 had similar insulin sensitivity, but reduced first-phase insulin response [6]. Further work will clarify how these subgroups, particularly group 3, relate to disruption of specific mechanistic pathways.
A large-scale data-driven approach to subclassify T2D was undertaken by Li et al. using electronic medical record data for 2551 individuals with T2D and 73 clinical features for topology-based patient-patient network generation [7]. Three distinct subgroups of T2D emerged, which appeared to have distinct additional disease risks and also unique genetic associations. Limiting the translatability of the findings, however, the study did not include replication of these subgroups in another dataset, and classification of a non-study patient into one of these three groups would not be straightforward. Additionally, it is unclear how to interpret the three groups in terms of underlying disease mechanism or relevance to patients in so far as actionability. Future work might also benefit from inclusion of ancestral background in the modeling approach to ensure that the specified subgroups do not simply represent categorization of patients by ancestry. Nevertheless, this work provided an exciting example of the potential of large-scale data and machine learning approaches to subtype complex disease.
Most recently, Ahlqvist et al. developed a new framework for characterizing adult onset diabetes based on six clinical metrics measured in Scandinavian individuals at the time of diabetes diagnosis: glutamic acid decarboxylase (GAD) antibody, age, body mass index (BMI), hemoglobin A1c, homeostatic model assessments of beta cell function (HOMA2-B), and insulin resistance (HOMA2-IR) [8••]. By applying k-means and hierarchical clustering algorithms, they identified five reproducible subgroups of patients: a severe autoimmune form (capturing T1D and LADA), a severe insulin-deficient form, severe insulinresistant form, mild obesity-related form, and mild agerelated form. The subgroups differed in terms of escalation of therapy and complications; for example, individuals in the severe autoimmune and severe insulin deficient clusters had the shortest times to sustained insulin use, and those in the severe insulin resistance cluster had the highest risk of developing chronic kidney disease. In a selected set of known T2D-associated genetic loci, at least one variant (rs7903146 in TCF7L2) had significantly different effects across the clusters, potentially supporting that the clusters are rooted in different biological disease processes [8••].
Applying the same data-driven approach as Ahlqvist et al. [8••], a subsequent study by Dennis et al. was performed using two large existing trial datasets of individuals with T2D randomized to metformin, sulfonylurea, or thiazolidinedione therapy [9]. The authors generated the same five clusters as previously observed in Ahlqvist et al. and found that these clusters differed in terms of glycemic progression, incidence of kidney disease, and glycemic response to medications. However, importantly, they noted that the observed differences between clusters could be better captured by other simple continuous features. For example, compared with analyses using the clusters, a model using only age at diagnosis similarly explained glycemic progression, and baseline-estimated glomerular filtration rate was a better predictor of time to chronic kidney disease. Similarly, a simple model incorporating sex, age at diagnosis, baseline BMI, and baseline HbA1c outperformed the clustering approach with regard to predicting treatment response. The authors therefore argue that "precision medicine in type 2 diabetes is likely to have the most clinical utility if it is based on an approach of using specific phenotypic measures to predict specific outcomes, rather than assigning patients to subgroups" [9].While this approach of using specific phenotypic measures for targeted clinical queries has pragmatic appeal, there may be benefits still to recognizing subcategories of disease, such as elucidation of underlying pathophysiology and development of novel targeted treatments.
Can the "Multiple Genes" Contributing to T2D Aid Subclassification?
Identification of subtypes of disease or clinical predictors rooted in mechanistic processes would lend themselves naturally to the promise of precision medicine: the notion that when a person's disease is not only well-characterized clinically, but also has a well-understood etiological basis, we can provide optimal, individualized management. T2D has a strong genetic basis, with heritability estimates ranging from 30 to 70% [10][11][12]. These heritability estimates capture genetic risk as well as shared familial, prenatal, and postnatal environmental exposures. Like other complex diseases, T2D is polygenic with thousands of germline genetic loci estimated to contribute to disease, including more than 200 that have been identified to date [13••]. Large-scale studies have helped shape our understanding of the genetic architecture of T2D, suggesting that common genetic variation is responsible for a significant proportion of disease risk and that causal variants at each locus are often non-coding, implicating a regulatory role [14••, 15••]. Given the non-coding nature of these genetic variants, it has been challenging to connect a given locus to specific regulatory elements, relevant gene(s), and tissue(s) [13••, 15••]; thus, translation of the hundreds of established T2D genetic loci into improved understanding of disease pathophysiology and clinical utility has been slow, leaving the potential of genomic medicine in diabetes currently unfulfilled. However, we are now in a time of unprecedented scale of genetic studies, access to large cohort and biobanks linking phenotype to genotype, and emerging technologies to interrogate genomics with highthroughput assays.
Nevertheless, a critical question to consider is whether genetics is relevant to T2D sub-classification. At this point in time, broadly across all complex diseases, the role of germline genetics (genetic variation that a person is born with and is present in essentially all cells of the body) in subclassification remains mostly speculative with few examples reaching patient care. It may be noted that genetics has found abundant clinical utility for complex disease in the realm of cancer, ushering highly effective targeted therapy; however, the gains achieved there have been largely using somatic genetic sequencing (capturing genetic variation that has occurred in specific cells during a person's lifetime) for molecular characterization of tumors to guide management [16]. Thus, given the limited precedent for clinical use of germline genetics for subtyping complex disease, it is worth reflecting on the lines of evidence supporting why genetics is not only relevant to T2D subclassification but also offers benefits beyond those seen with solely phenotype-based approaches.
First, the utility of genetics in T2D subclassification is supported by the existence of monogenic diseases that are frequently misdiagnosed as T2D. Indeed, dozens of genes have been implicated in monogenic diabetes. These conditions present clinically either with diabetes being the predominant disease feature, as is seen with forms of maturity onset diabetes of the young (MODY) and neonatal diabetes, or with diabetes existing as part of a syndromic presentation, as with mitochondrial diabetes and Wolfram syndrome. The genotype-phenotype relationships for several monogenic diabetes conditions were recently reviewed in guidelines published by the International Society for Pediatric and Adolescent Diabetes (ISPAD) [17••]. Monogenic diabetes collectively accounts for at least 0.4% of all diabetes cases, with MODY being the most common type [17••, 18]. It has been estimated that close to 80% of individuals with MODY remain undiagnosed [19]; many are living with a misdiagnosis of T2D, since the age of onset and clinical features of patients with MODY can overlap with T2D. Of course, the single pathogenic variants conferring large disease risk seen in monogenic diabetes differ considerably from the common genetic variation associated with small incremental risk of T2D; however, the fact that patients with MODY are frequently misdiagnosed with T2D highlights that employing genetics to improve detection of MODY (and other forms of monogenic diabetes) would reduce the heterogeneity of T2D (and T1D) attributable to misdiagnosed monogenic disease.
Second, and related to the first point, is that even within categories of phenotypically defined disease subgroups, there may exist genetic heterogeneity that is clinically important. There are numerous examples across medicine of so-called phenocopies, diseases with indistinguishable phenotypic characteristics, but with distinct genetic causes (e.g., multiple endocrine neoplasia (MEN) types 1 and 4). Within diabetes, MODY provides an illustrative example: patients who are clinically similar in terms of gross phenotypic and biochemical features (i.e., normal to low BMI, diabetes onset before age 30, preserved beta-cell function, and without evidence of autoimmunity) may have monogenic diabetes caused by several different genes, including most commonly GCK, HNF1A, and HNF4A. While in expert hands, patients with different genetic forms of MODY may be distinguishable based on careful, deep phenotyping, a genetic diagnosis provides objective evidence of distinct disease entities that can be phenotypically similar. Furthermore, in the case of MODY, we are fortunate to have knowledge that the implicated genetic alteration can guide management (e.g., patients with GCK MODY are generally safe without diabetes treatment and those with HNF1A/HNF4A can often be transitioned from insulin to oral therapy with sulfonylureas) [18]. While we are concerned with polygenic risk in T2D (rather than monogenic risk seen in MODY), clinically relevant genetic heterogeneity may also exist within T2D disease subtypes that are clinically indistinguishable. In these situations, it is possible that our current inability to recognize heterogeneity within clinically similar appearing individuals is due in part to relevant biomarkers being unknown or unavailable, and genetics offers an agnostic as well as relatively holistic diagnostic approach.
Third, genetics may guide disease management. In contrast to clinically defined subtype, a genetically defined subtype of disease lends itself more readily to potential mechanism-focused management strategies. Again, it is important to note that single genetic perturbations associated with monogenic disease will typically impose greater downstream consequences and impact on disease risk than those associated with T2D (which have modest effect sizes, generally OR < 1.2). However, it is possible that there will be individuals in whom composite polygenic risk impacts one or more specific disease pathways profoundly, and thus, such pathways could be useful for targeting management [20].
Finally, in contrast to many clinical and laboratory features, germline genetic markers do not change throughout a lifetime and are unaffected by treatments or disease course. Thus, a test assessing genetic classification could be applied at any time during disease course, including years after initial diagnosis and medication initiation or even potentially prior to diagnosis such that a preventative strategy could be employed.
In particular, the evolving story of the TM6SF2 association with T2D provides an elegant example of genetics enabling discovery of a novel T2D disease mechanism. The CILP2 locus containing multiple genes, including TM6SF2, was initially associated with T2D in a meta-analysis of T2D genomewide association studies (GWAS) published in 2012 [27]. Subsequently, exome chip analysis containing proteincoding variants across the genome identified an amino acidaltering variant in TM6SF2 p.Glu167Lys as significantly associated with T2D, and two variants resulting in amino acid substitutions in this gene (p.Glu167Lys and p.Leu156Pro) were driving the locus association signal [15••]. The same variant TM6SF2 p.Glu167Lys was significantly associated with non-alcoholic fatty liver disease (NAFLD) in an independent exome-chip analysis [25]. The shared T2D-and NAFLDincreasing allele of this variant was also associated with higher circulating levels of alanine transaminase, a marker of liver injury, and with lower levels of triglycerides [25]. Functional experiments knocking down Tm6sf2 in mice [25] as well as TM6SF2 in human hepatoma cell lines [26] both supported that reduced gene expression increased liver triglyceride content and decreased secretion of triglyceride-rich lipoproteins from liver tissue, thus implicating TM6SF2 in liver fat metabolism. These exciting findings provided a novel disease pathway of primary liver tissue origin leading to increased risk of both T2D and NAFLD, demonstrating the power of genetics to uncover disease mechanism.
While a growing number of causal genes have been confidently connected to GWAS loci, the majority of T2D loci remain unmapped [13••]. As an alternative strategy to connect GWAS loci to mechanistic pathways, recent studies, including work from our lab, have leveraged multiple variant-trait associations to generate groups of related genetic variants and subsequently infer disease pathways [15••, 28-30]. As mentioned previously, a major challenge in connecting GWAS loci to relevant pathways is that the loci include dozens of variants highly correlated with one another, most of which are non-coding.
Thus, despite advances in fine-scale mapping [13••, 15••, 31], for most T2D loci, we currently do not know which genetic variants, regulatory elements, genes, or tissues are functionally relevant. Multi-trait cluster analysis offers an opportunity to bypass this missing information and connect variants directly to pathways via the pattern of trait associations. In this approach, each T2D locus is represented by a variant associated with T2D at genome-wide significance, and the diabetes-riskincreasing allele is interrogated for its effect on multiple different traits. A clustering approach can then be applied to group variants together which share similar patterns of associations across multiple traits. The two most recent clustering efforts [15••, 29••] have found that applying a "soft" clustering approach, where variants can belong to one or more clusters, rather than a "hard" clustering approach [28,30], which requires that each variant only belong to one cluster, produced more readily interpretable results. The "soft" clustering approach appears to be well-suited for modeling complex disease biology, since it allows a given locus to impact one or more genes, which in turn may alter one or more disease pathways.
In Udler et al., clustering of 94 T2D variants and 47 metabolic traits using the soft clustering approach Bayesian nonnegative matrix factorization (bNMF) produced five groupings of genetic loci (Fig. 1), each with distinct tissue-specific enhancer and promoter enrichment based on analysis of epigenomic data from 28 cell types [29••]. Two clusters contained variant-trait associations indicative of reduced betacell function, differing from each other by high vs. low proinsulin levels, suspected to represent defective insulin processing vs. defective insulin synthesis, respectively. The three other clusters of loci represent mechanisms of insulin resistance: obesity-mediated (high BMI and waist circumference), "lipodystrophy-like" fat distribution (low BMI, adiponectin, and HDL-cholesterol, and high triglycerides), and disrupted liver lipid metabolism (low-serum triglycerides). In line with the prior discussion of functional work supporting the role of TM6SF2 in liver fat metabolism, this locus was most strongly weighted in the final liver lipid metabolism cluster. The clusters were enriched for active regulatory elements in tissues which were consistent with suspected pathways; for example, the defective insulin processing cluster was most strongly enriched for regulatory elements in pancreatic islet cells compared with the other 27 cell types (P < 0.001), and the disrupted liver lipid metabolism cluster was significantly enriched for elements in liver tissue (P < 0.001). The epigenomic data thus provided additional support that these genetically informed pathways Fig. 1 Based on the "Hallmarks of Cancer" presented in a landmark cancer biology paper by Hanahan and Weinberg [32], the different disease pathways contributing to diabetes are presented as the "Hallmarks of Diabetes." Pathways are separated into mechanisms leading either to insulin deficiency or insulin resistance; insulinindependent mechanisms are not included here. The question marks indicate that additional disease pathways are yet to be identified represent distinct disease mechanisms [29••]. Additionally, the loci implicated in each cluster were differentially associated with other metabolic diseases: the defective insulin processing loci T2D-increasing alleles were most strongly associated with stroke risk and the lipodystrophy loci T2D-increasing alleles were most associated with increased systolic and diastolic blood pressure [29••].
Using another "soft" clustering approach called C-means, Mahajan et al. similarly analyzed GWAS data for a set of 94 T2D association signals partly overlapping with those used in Udler et al. [29••] and 10 T2D-related quantitative traits, identifying six variant clusters (shown in Supplementary Fig. 6b of [15••]). Five of the clusters broadly mapped to the same clusters from Udler et al. described above. Thus, reassuringly, two independent approaches of clustering T2D variant-trait associations resulted in largely similar findings, suggesting robustness in these genetically driven disease pathways.
In considering the role of genetics in elucidating disease mechanisms, a conceptual model proposed for cancer biology may be useful for diabetes biology. A landmark cancer biology paper by Hanahan and Weinberg describing the pivotal deregulated pathways underlying cancer development offers a framework to deconstruct the intricacies of this complex disease [32]. These functional pathways, termed the "Hallmarks of Cancer," can perhaps inspire us to conceptualize the "Hallmarks of Diabetes" (Fig. 1), and consider how defining key diabetes pathways may likewise eventually guide disease classification and management. In the case of cancer, perturbations of these pathways (typically via somatic genetic alteration, potentially on a germline risk background) induce tumor development and sustained growth. Different cancers may display deregulation within specific functional pathways, highlighting disease etiology and guiding treatment selection. For example, tumors displaying deregulated apoptotic pathways are often responsive to drugs that induce cancer cell apoptosis [33]; tumors with defective DNA damage repair mechanisms may respond well to immune-directed therapies [34] or drugs that exploit genomic instability [35]; and cancers with abnormal signaling pathways can respond to kinase inhibitors [36]. Therefore, identification of key functional pathways within such a framework can reduce disease complexity, improve understanding of disease mechanism, and inform the development and clinical use of targeted therapies. By all means, cancer and diabetes disease biology have important differences, and the discussion presented here of diabetes biology is restricted to germline variation; however, cancer serves as a powerful example of how a holistic approach to defining pathway deregulation can be integrated with our understanding of genomic variation to transform the treatment of disease. As we continue to identify the pathways, or hallmarks, of diabetes, we can start to ask whether individuals develop disease predominantly through one or multiple pathways and whether these pathways can be targeted for treatment. Moreover, if in fact most individuals develop diabetes via deregulation of multiple pathways, as is suspected [20], a "poly-pill" combination therapy that targets multiple pathways could be beneficial.
As underlying disease mechanisms become elucidated, our conceptualization of metabolic disease will expand. T2D is, in a sense, an artificial construct based on a threshold chosen against the continuum of hyperglycemia. Likewise, other metabolic diseases, such as obesity and hypertension, are based on chosen thresholds for continuous traits (BMI and blood pressure measurements). The cluster-defined pathways leading to increased T2D risk also appear to impact risk of other metabolic conditions, raising the notion that shared disease processes or "endophenotypes" may underlie metabolic diseases (Fig. 2). As our knowledge of genetic variation impacting metabolic diseases continues to grow, clustering and other approaches will continue to refine our understanding of these critical endophenotypes, such as dysregulated insulin processing or unfavorable fat distribution. Appreciating the molecular basis for shared risk among metabolic diseases might help improve disease screening among unaffected individuals and also screening and/or preventative steps to reduce complications among those with T2D.
Efforts to Use Genetics to Subtype T2D's "Many Diseases" Identification of mechanistic disease pathways causing T2D will no doubt be useful for improved understanding of pathophysiology and drug development. Beyond these benefits, a natural next question to ask is whether genetically defined clusters of loci can identify subtypes of diabetes. Genotype information could be used to identify individuals carrying many alleles within a given cluster, potentially representing multiple "hits" along a pathway. In this scenario, diabetes in these individuals would be driven predominantly by one or few pathways, and knowledge of the underlying disease pathway(s) could guide management.
At this point in time, such a genetic cluster-based approach to diabetes classification is not ready for application in the clinic. Our group has shown, however, that genetically defined subgroups of T2D can identify individuals with differing phenotypic traits, thereby serving as an initial step in deconstructing the heterogeneity of T2D [29••]. In Udler et al., pathway-specific genetic risk scores (GRS's) for the five genetic clusters were generated for 17,365 individuals with T2D, combined across four cohorts, to determine whether individuals with the top 10% GRS uniquely for each cluster would have clinical differences. The cut-off of top 10% was arbitrarily chosen, and further work is needed to set more clinically relevant thresholds; however, differences between subgroups were observed using this 10% ("extreme") threshold. Indeed, those patients with T2D with extreme GRS for the two insulin deficiency clusters had lower C-peptide levels than all other individuals with T2D (P < 0.01, combined); those with extreme obesity cluster GRS had significantly increased BMI, percent body fat, hip circumference, and waist circumference (P values < 0.05); those with extreme lipodystrophy cluster GRS had significantly decreased highdensity lipoprotein cholesterol, percent body fat, and BMI (P values < 0.01); and those with extreme liver lipid metabolism cluster GRS had significantly decreased serum triglyceride levels (P = 0.01). Thus, individuals with T2D and a GRS uniquely at the top 10% of one cluster had representative trait characteristics collectively distinguishing them from all other individuals with T2D. Further work is underway to determine whether those at the highest percentile of a cluster respond differentially to any medications or are at differential risk for complications of diabetes.
Clinically relevant genetic subtyping for T2D might also involve utilizing a GRS developed for predicting T1D risk. In its most updated form, the 67-SNP T1D GRS was highly discriminative for identifying individuals with T1D (area under the receiver operator characteristics curve (AUC) of 0.92) [37]. Additionally, a T1D GRS may have utility in identifying individuals with later age onset T1D who are misdiagnosed as T2D [38] as well as predicting escalation to insulin therapy in patients with presumed T2D with GAD Ab positivity [39]. In the latter study, a 30-SNP T1D GRS was calculated in 8608 individuals diagnosed with T2D after 35 years of age and treated without insulin for at least 6 months following diagnosis. The T1D GRS predicted progression to insulin use at five years, but only in GAD-positive participants: probability of insulin use (95% CI): 47.9% for high T1D GRS (35.0%, 62.78%) vs. 27.6% for medium T1D GRS (20.5%, 36.5%) vs. 17.6% for low-risk T1D GRS (11.2%, 27.2%); P = 0.001 [39]. Interestingly, there was no association with insulin use at 5 years in GAD-negative individuals, which comprised the majority of study participants (96.7%). One can imagine that eventually, a more intricate model including T2D pathwayspecific GRS as well as T1D pathway-specific GRS may be useful for improved modeling of diabetes subgroups.
Conclusion
Exciting progress has been made in exploring approaches to "slice up" the large, approximately 90%, T2D portion of the diabetes subtype pie. The clinically defined subgroups proposed by Ahlqvist et al. [8••] will no doubt undergo deeper Fig. 2 A future holistic vision for genetic variation contributing to metabolic conditions via disease processes, termed endophenotypes. Genetic variants shown at the bottom of the figure within DNA colored in red contribute to multiple endophenotypes whereas those in pink contribute to only one. It is hypothesized that across multiple metabolic diseases, thousands of distinct genetic signals contribute to dozens of endophenotypes. The endophenotypes represent targets for therapy and importantly targeting a given endophenotype may affect one or more metabolic disease conditions physiological and genetic characterization in the coming months, potentially leading to increased appreciation of the underlying mechanistic processes for each cluster. Additionally, with increased awareness of monogenic diabetes and access to genetic testing, the small slice of misdiagnosed monogenic diabetes cases will be chipped away from the T2D segment of the pie. It is likely that pathway-specific genetic GRSs will be further developed with clinically relevant thresholds determined, such that these "hallmarks of diabetes" can be utilized to further chip away at the T2D segment and guide management.
Challenges will remain. For example, placing a new individual into clinically derived clusters, such as those developed by Ahlqvist et al. [8••], is not straightforward in practice, particularly if distributions of the various clinical factors differ across populations (e.g., distributions of BMI or HbA1c may differ in patients with diabetes in Scandinavia compared with those in the USA). Applying the method to populations of different ancestries and development of online calculators will facilitate clinical translation. Pathway-specific genetic risk scores, likewise, have been developed based on data from individuals of European ancestry, and availability of GWAS summary statistics for analyses in diverse ancestral populations will be necessary for GRSs to be developed for individuals of non-European ancestry.
Finally, perhaps new approaches will be developed that combine clinical and genetic characteristics into a single model, ideally also incorporating environmental factors such as dietary and lifestyle habits. While inclusion of genetics into classification schemas will require additional research, it has the potential to improve our understanding and management of T2D, both as a single disease and as a part of an integrated metabolic disease network. | 2019-07-11T13:13:57.612Z | 2019-07-10T00:00:00.000 | {
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49291210 | pes2o/s2orc | v3-fos-license | Reemergence of Reston ebolavirus in Cynomolgus Monkeys, the Philippines, 2015
In August 2015, a nonhuman primate facility south of Manila, the Philippines, noted unusual deaths of 6 cynomolgus monkeys (Macaca fascicularis), characterized by generalized rashes, inappetence, or sudden death. We identified Reston ebolavirus (RESTV) infection in monkeys by using serologic and molecular assays. We isolated viruses in tissues from infected monkeys and determined viral genome sequences. RESTV found in the 2015 outbreak is genetically closer to 1 of the 4 RESTVs that caused the 2008 outbreak among swine. Eight macaques, including 2 also infected with RESTV, tested positive for measles. Concurrently, the measles virus was circulating throughout the Philippines, indicating that the infection of the macaques may be a reverse zoonosis. Improved biosecurity measures will minimize the public health risk, as well as limit the introduction of disease and vectors.
R eston ebolavirus (RESTV) was discovered after an outbreak of hemorrhagic disease in cynomolgus macaques in a primate research facility in Reston, Virginia, USA in 1989 that had imported macaques from the Philippines (1). Subclinical infections in humans in the facility were determined through diagnostic testing. Other outbreaks of RESTV epizootics were identified in Sienna, Italy in 1992 (2); Alice, Texas, USA in 1993 (3); and 2 outbreaks in the Philippines in 1996; all 4 outbreaks involved purpose-bred cynomolgus macaques (Macaca fascicularis) attributed to a single nonhuman primate (NHP) facility in the Philippines (4). Until the 2015 outbreak described here, no outbreaks of RESTV had occurred in the Philippines since 1997; subsequently, the government permanently closed the facility.
The last known occurrence of RESTV epizootic in the Philippines was during 2008-2009 and affected 2 piggeries on the island of Luzon, 1 of the 3 major islands in the country. The disease was discovered as a co-infection with porcine reproductive and respiratory syndrome virus (PRRSV), also prevalent at that time (5). After this outbreak, Jayme et al. undertook a search for a possible reservoir in bats by using low levels of viral RNA detected in the microbat Miniopterus schreibersii (6).
As part of the established process for testing of macaques in the quarantine facility, animals that are sick or die are routinely tested for the presence of RESTV infection. In August 2015, six monkeys that were in the last stage of quarantine died suddenly; their bodies were submitted for testing. Although there are many fruit-bearing trees in the facility, the building was constructed in such a way that fruit bats could not make contact with the monkeys. However, rats were observed entering the cages of individual primates in the facility.
At the same time, an outbreak of measles virus (MV) was occurring among humans nationwide. During the first 6 months of 2015, there were 2,231 reported cases, of which 534 were laboratory-confirmed (7).
In this study, we describe the serologic and molecular detection of RESTV and MV from macaques in the quarantine facility in the Philippines in 2015, and demonstrate genetic characterization of the isolated RESTV.
Facility
The size of the monkey quarantine facility, located in the province of Southern Luzon, is ≈3,000 km 2 . The 174 monkeys sampled were housed in 2 separate buildings that are equipped with individual stainless steel squeeze cages measuring ≈58 × 48 × 77 cm 3 , arranged in 4 rows: the cages in the first and second rows, and those in the third and fourth
Reemergence of Reston ebolavirus
in Cynomolgus Monkeys, the Philippines, 2015 rows face each other. Each building has its own anteroom and is surrounded by large windows that have screens and welded wire to protect the monkeys from vermin and prevent monkey escapes. Each cage is equipped with a lock and squeeze-back mechanism. There were separate personnel assigned to each building and each were required to wear individual personal protective equipment (undergarments, coveralls, mask, caps, goggles, socks, and boots) and to shower when entering and exiting the animal buildings. Materials, such as those used during animal care procedures, as well as cleaning implements, were not shared between buildings. We used new sterile disposable syringes with needles for each monkey and for each procedure. We disposed of used syringes and needles in dedicated containers in each building and disposed of them through a government-accredited waste contractor.
Samples
Both antigen and antibody detection methods were used in the laboratory investigation of the epizootic occurrence.
We collected a total of 174 samples from the facility for RESTV IgG and MV IgM screening. Blood samples were centrifuged on site and serum samples were transferred to labeled cryovials and transported through a cold chain. The serum samples were heat-inactivated at 56°C for 1 h. Spleen, liver, and lymph nodes from 4 deceased monkeys were also collected and transported in the same manner as the sera and tested for RESTV by using molecular assays. Serum samples from macaques in the 2 breeding facilities located in Oriental Mindoro and Rizal that supplied the macaques to the quarantine facility were also tested for RESTV antibodies, as were 71 personnel in the facilities.
RESTV Serologic Analysis
Indirect ELISA testing was used following the protocols of either the Centers for Disease Control and Prevention (CDC) (8) or the protocol of the National Institute of Infectious Diseases (Musashimurayama, Tokyo, Japan) reagents (9). Briefly, for the CDC protocol, the upper half of the ELISA plate (Falcon, Avenel, NJ, USA) was coated with gamma-irradiated antigens obtained from a RESTVinfected cell suspension, and the lower half with those obtained from a non-infected cell suspension. We added test samples to the wells, diluted 4-fold starting at 1:100. We used mouse anti-human IgG with horseradish peroxidase (Accurate Chemical and Scientific Company, Westbury, NY, USA), diluted at 1:4,000, as a secondary antibody. We added 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid substrate (Kirkegaard-Perry Laboratories, Gaithersburg, MD, USA) at the last step for visualization of the antigen-antibody reaction. The optical density value was recorded at 415 nm by using an ELISA plate reader (ThermoFisher Scientific, Carlsbad, CA, USA).
Samples were considered reactive if the adjusted optical density (OD) was >0.95. For the National Institute of Infectious Diseases protocol, the upper half of the ELISA plate was coated with RESTV recombinant nucleoprotein (NP) tagged with glutathione S transferase, expressed in Escherichia coli at ≈100 ng/well, and the lower half with negative control glutathione S transferase antigen. Goat anti-human IgG conjugated with Novex horseradish peroxidase (ThermoFisher) diluted at 2 μg/mL was used as a secondary antibody. Samples were considered reactive if the sample showed an OD >0.56 at 1:100 dilution, or 0.23 at 1:400 dilution.
We retested all serologically reactive samples by using immunofluorescent assay (IFA) as described by Ikegami et al. (10). In brief, serum samples were 2-fold serially diluted in phosphate-buffered saline (PBS) from 1:10 to 1:640. Diluted serum (20 µL) was loaded onto each well of the IFA slide (14 wells; AR Brown Co., Ltd., Toyo, Japan) containing HeLa cells expressing RESTV recombinant NP. The slides were incubated for 1 h at 37°C and washed 3 times with PBS. Invitrogen Fluorescein isothiocyanatelabeled antibody against human IgG (ThermoFisher) diluted in PBS at 1:200 was added to each well and incubated for 1 h at 37°C. After washing with PBS, the slides were examined for the staining pattern under an immunofluorescent microscope (Nikon, Chiyoda, Japan) and their reactions were recorded.
In addition, we tested all reactive serum samples for antibodies against RESTV glycoprotein in a Luminex assay (Luminex Corporation, Austin, TX, USA). Briefly, Luminex beads coated with RESTV glycoprotein (Bead #35) were blocked in 2% skim milk Tween and phosphatebuffered saline (TPBS) for 30 min at room temperature in the dark with shaking in a flat-bottom microtiter plate. We washed the plate twice with TPBS by using a magnetic plate washer (BioPlex Pro II Wash Station; Bio-Rad, Hercules, CA, USA). Serum diluted (1:100, 100 µL) in TPBS was added and incubated for 30 minutes, as stated before. The plate was washed and 100 µL of a mixture of biotinylated Protein A (1:500)/biotinylated Protein G (1:250) (ThermoFisher Scientific, Brisbane, Queensland, Australia) was added to each well and incubated as described above. The plate was washed again, then 100 µL of streptavidin-phycoerythrin was added (1:1,000; Thermo Scientific, Brisbane, Queensland, Australia), and the plate was incubated as before. Samples were assayed on the BioPlex machine (Bio-Rad) and the median fluorescence intensity read for 100 beads.
Measles Serologic Analysis
The 174 serum samples from macaques in the primate quarantine facility were tested for MV antibody by Enzygnost Measles Anti-IgM ELISA (Siemens Healthcare Diagnostics, Marburg, Hesse, Germany). We diluted the serum samples in rheumatoid factor adsorbent to remove inhibitors that might interfere with the reaction. We used the ELISA by following the manufacturer's instructions. A sample was determined to be negative if the corrected OD at a wavelength of 450 nm with a reference at a wavelength of 635 nm was <0.100, equivocal if it was 0.101-0.199, and positive for MV IgM if it was >0.200.
Molecular Detection of RESTV
We isolated total RNA from lymph node, liver, and spleen samples of infected animals by using a combined method of TRIzol inactivation and QIAGEN RNeasy Mini Kit (QIAGEN, Copenhagen, Denmark) RNA isolation as prescribed by CDC (11). We amplified the RESTV viral genome by targeting the NP gene developed by Sanchez et al. (12). The reaction was completed in a 20-µL reaction mixture containing 4 µL of RNA, 0.2 mmol/L of each dNTP, 1.6 mmol/LM MgSO 4 , 0.8 µL of Super-Script III RT/Platinum Taq enzyme mix (Invitrogen), and 0.5 µmol/L each of the primers RES-NP1 and RES-NP2. Thermocycling conditions were set to RT at 50°C for 30 min, inactivation and initial denaturation at 95°C for 3 min, then 45 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, then ending with a final extension step of 72°C for 5 min. Visualization of DNA bands was performed following electrophoresis in 2% agarose gels. Amplification of the latent gene from tissues was also undertaken by using the method adapted from Zhai et al. (13) using the Superscript Platinum Taq One-Step RT-PCR kit to generate 611-bp PCR products.
We then purified the PCR products and sequenced them with the BigDye Terminator v3.1 (ThermoFisher Scientific, Waltham, MA, USA) and analyzed in a 3500 Series Genetic Analyzer (Applied Biosystems, Foster City, CA). Assembled sequencing results were subjected to BLAST alignment (https://www.ncbi.nlm.nih.gov/BLAST), which confirmed the identity of the sequences as RESTV.
Molecular Detection of Measles Virus
A TaqMan RT-PCR assay, distributed by CDC (14), was performed to detect MV RNA from macaques in the quarantine facility. Amplification of the MV RNA from sample DrpZ2-10B-G was undertaken using the HEN_RES_MOR primers described by Tong et al. (15) and produced a product of the expected size, which was sequenced.
RESTV Isolation from Tissues
We attempted virus isolation in the liver; spleen; axillary lymph nodes (from DrpZ1-103A-K, DrpZ5-2B-F, and DrpL7-7D-A), and axillary, cervical, inguinal, and mesenteric lymph nodes (from DrpZ2-10B-G). Tissues were homogenized in PBS for 30 s with silicon carbide beads, centrifuged for 5 min, and the supernatant was added to a flask of semiconfluent Vero C1008 cells in PC4 containment at the CSIRO Australia Animal Health Laboratory, Victoria, Melbourne, Australia. For detection of virus replication, we performed 3 blind passages, monitored for CPE, and used a real-time PCR specific for NP of RESTV (J.S. Towner and P. Rollin, US Centers for Disease Control and Prevention, pers. comm., 2009 May 1).
Next-Generation Sequencing of Isolates
We performed next-generation sequencing on the supernatant from passage 2 of the isolates from the axillary lymph nodes following TRIzol purification of RNA by using Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA). RNA was transcribed to cDNA by using random primers and SuperScript III reverse transcription (Thermo-Fisher SuperScript III First-strand Synthesis System), and then cDNA was prepared with Klenow (Promega, Madison, WI, USA). We used the Illumina Nextera XT library preparation kit to prepare dual barcoded libraries for 150bp paired-end sequencing on the MiSeq system (Illumina, Scoresby, Victoria, Australia).
Serologic Analysis
The RESTV ELISA showed that 10 of 174 samples (5.74%) from macaques were reactive for RESTV IgG antibodies ( Table 1), all of which had an IFA titer >640 (Figure 2). These samples were also reactive in a Luminex assay, confirming the presence of IgG against RESTV. Of the 174 serum samples, 8 (4.59%) were reactive for MV IgM in the ELISA; 1 of the macaques (Identification no. DrpZ1-26D-B) was serologically positive for RESTV and MV antibodies (Table 1). Among those serologically positive for only MV IgM, 1 macaque (identification no.: DrpZ2-10B-G) showed a positive result for a RESTV PCR in an autopsy sample ( Table 2). The data indicated that among 174 macaques, 2 (1.1%) had a history of infection with RESTV and MV.
Of the macaques from the 2 breeding facilities located in Oriental Mindoro and Rizal that supplied the animals to the quarantine facility, 10% were tested, as were personnel from both facilities. None of these personnel or macaques had detectable antibodies to RESTV.
Virus Isolation and Molecular Analyses
RESTV was successfully isolated in Vero C1008 cells from the inguinal and axillary lymph nodes of DrpZ5-2B-F and the axillary lymph node of DrpZ2-10B-G (Table 2). Initial amplification of a 337-bp product of the partial NP gene from the liver, spleen, and lymph node tissue samples in 4 NHPs confirmed the presence of RESTV. Further amplification and sequencing of the partial L gene along with realtime detection further confirmed the RESTV infection. In all cases, Blast N revealed that the RESTV in this outbreak 1288 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 24, No. 7, July 2018 was most similar to the virus from the 2008 outbreak in swine (GenBank accession no. FJ624585.1) rather than the 1996 outbreak in NHPs. The comparison of the whole genome sequencing of the 2 isolates DrpZ5-2B-F and DrpZ2-10B-G showed that there were 3 nucleotide differences. The first variation noted was in the NP gene (position 837 of the genome) of DrpZ5-2B-F, which resulted in a non-conservative amino acid change of a Thr (ACG) to a Lys (AAG) when compared with DrpZ2-10B-G and other RESTV isolates ( Table 3). The change was also observed for all 4 specimen types of DrpZ5-2B-F (15-009-012), confirming that the change was not caused by passaging of the virus in cell culture. The second change noted was at position 4393 of the genome in the noncoding region between virus capsid proteins 35 and 40, and resulted in an adenosine for DrpZ5-2B-F and a guanine for DrpZ2-10B-G. The third variation was at position 10787 of the genome, and resulted in an amino acid change at position 162 of the VP24 protein from an Asn (AAC), which is common to all RESTV strains, to a Lys (AAA) for DrpZ5-2B-F. Therefore, DrpZ5-2B-F had 2 unique changes compared with other RESTV isolates. Both isolates showed 98% similarity to their closest RESTV strain (GenBank accession no. FJ621585.1).
Because among 4 macaques that had a positive result by RESTV PCR, 1 (ID: DrpZ2-10B-G) was serologically positive for MV (Table 1), we subjected a lymph node sample of this macaque to testing for MV by TaqMan RT-PCR to confirm the dual infection. As a result, we detected the MV genome, indicating that a dual infection occurred in this macaque ( Table 2). Amplification of the partial L gene of MV RNA from sample DrpZ2-10B-G, followed by sequencing of the product and a BlastN of the sequence, revealed that the MV belonged to genotype B3, which had caused a large outbreak in the Philippines in 2014 (16) (Figure 3). MV RNA was also detected in 6 other macaques in the quarantine facility by using Taq-Man RT-PCR.
Discussion
In 2015, 19 years after the last known epizootic occurrence of RESTV in macaques in the Philippines, we detected and confirmed the incidence of RESTV in macaques in a primate facility south of Manila, by serologic and molecular testing. In spite of the long hiatus, RESTV was found in a controlled environment in which monkeys are systematically housed to avoid spread of diseases and to which no wild monkeys have been introduced. Personnel in the facility had no evidence of infection because no RESTV antibodies were detected.
We observed rats in cages in the primate facility that housed the primates being tested, indicating the potential for small animals to gain access to the facility. A recent study identified the microbat Miniopterus schreibersii as a possible reservoir of RESTV (6); therefore, this bat species and similar ones of this size may be the source of infection in the quarantine facility. If this is the case, improved biosecurity measures are warranted to limit the introduction of disease. However, we do not claim the bat species as the direct source of infection in 2015 outbreak. Because the facility building has its own anteroom with welded wire window screens, there is little likelihood that bats entered the facility.
Dual infections of RESTV and simian hemorrhagic fever virus (SHFV) in cynomologus monkeys have been reported in a facility in Reston, Virginia, and SHFV is the suspected causal agent for mortality in monkeys (17). Dual infections of RESTV and PRRSV in swine have been identified in the Philippines (5) and in Shanghai, China (18). In these cases, all of the RESTV-positive swine were coinfected with PRRSV. In contrast, we found in this study that 1 (ID: DrpZ1-26D-B) of the 10 macaques positive for RESTV antibody was also positive for MV antibody. Furthermore, another macaque (ID: DrpZ2-10B-G) was confirmed to have dual infection of RESTV and MV by using PCR. The results show similarities with dual infections such as SHFV and RESTV in macaques (17), or RESTV and PRRSV infections in swine (5). However, MV was not detected among most macaques positive for RESTV that died from the disease. Also, it remains unclear whether the MV infection supports an increase in RESTV replication in macaques. We found that 8 macaques had antibodies against MV, and 1 was MV PCR positive. Considering the risk for humanto-primate transmission (19,20), there is a possibility that MV infection in macaques is associated with human MV outbreak in the Philippines, although further studies are required to identify the mode of transmission of MV infection in macaques. The RESTV sequences obtained were most similar to Reston-08-E from the Philippines 2008 outbreak in swine (5) (Figure 1). There were 3 nucleotide variations between the viral isolates that were sequenced, 2 of which in isolate DrpZ5-2B-F resulted in nonconservative changes in the NP and VP24 proteins that were unique when compared to all of the RESTV isolates sequenced. Because of the similarity with other Ebola viruses and the virus' ability to infect humans, there is a concern that RESTV could mutate during passage through animals like macaques and cause an epidemic of disease in humans. Because it could mutate to pose health consequences for humans, continued surveillance is required to reduce the risk of transmitting Reston Ebola virus. | 2018-06-25T07:47:15.222Z | 2018-07-01T00:00:00.000 | {
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54936071 | pes2o/s2orc | v3-fos-license | Habitability of the Goldilocks Planet Gliese 581g: Results from Geodynamic Models
Aims: In 2010, detailed observations have been published that seem to indicate another super-Earth planet in the system of Gliese 581 located in the midst of the stellar climatological habitable zone. The mass of the planet, known as Gl 581g, has been estimated to be between 3.1 and 4.3 Earth masses. In this study, we investigate the habitability of Gl 581g based on a previously used concept that explores its long-term possibility of photosynthetic biomass production, which has already been used to gauge the principal possibility of life regarding the super-Earths Gl 581c and Gl 581d. Methods: A thermal evolution model for super-Earths is used to calculate the sources and sinks of atmospheric carbon dioxide. The habitable zone is determined by the limits of photosynthetic biological productivity on the planetary surface. Models with different ratios of land / ocean coverage are pursued. Results: The maximum time span for habitable conditions is attained for water worlds at a position of about 0.14+/-0.015 AU, which deviates by just a few percent (depending on the adopted stellar luminosity) from the actual position of Gl 581g, an estimate that does however not reflect systematic uncertainties inherent in our model. Therefore, in the framework of our model an almost perfect Goldilock position is realized. The existence of habitability is found to critically depend on the relative planetary continental area, lending a considerable advantage to the possibility of life if Gl 581g's ocean coverage is relatively high. Conclusions: Our results are a further step toward identifying the possibility of life beyond the Solar System, especially concerning super-Earth planets, which appear to be more abundant than previously surmised.
Introduction
The nearby M-type dwarf Gliese 581 is an important target of previous and ongoing planetary search efforts, and so far four, but possibly up to six planets around this star have been discovered. It is noteworthy that until recently the possible existence of Earth-type and super-Earth planets in extrasolar systems has been highly speculative (e.g., Butler et al. 2006). Bonfils et al. (2005) reported the detection of a Neptune-size planet around Gl 581 (M3 V). Gl 581 is at a distance of 6.26 pc; it has a mass of about 0.31 M ⊙ and a luminosity of 0.013 ± 0.002 L ⊙ (see below).
Later on, Udry et al. (2007) announced the detection of two so-called "super-Earth" planets in this system, Gl 581c and Gl 581d, with minimum masses of 5.06 and 8.3 M ⊕ and with semi-major axes of 0.073 and 0.25 AU, respectively. In 2009, Gl 581e was discovered, another super-Earth planet (Mayor et al. 2009). Meanwhile the position of Gl 581d has been re-identified as 0.22 AU. Finally, in 2010, Vogt et al. (2010) reported a still controversial discovery. The authors argue in favor of the existence of two additional super-Earth planets, namely Gl 581f and Gl 581g. Note that Gl 581g is of particular interest to the study of planetary science and Send offprint requests to: W. von Bloh astrobiology because it is positioned in the midst of the stellar climatological habitable zone. Its mass is estimated to be between 3.1 and 4.3 M ⊕ , noting that the putative upper mass limit is partially constrained by the assumption that none of the planetary orbital eccentricities exceeds 0.2. The distance of Gl 581g is estimated to be 0.14601±0.00014 AU, and its eccentricity is almost identical to zero.
According to Valencia et al. (2006), super-Earths are rocky planets from one to about ten Earth masses with a chemical and mineral composition akin to that of Earth. In the following, we adopt the hypothesis that this is indeed the case, and consider for Gl 581g our model previously developed for the Earth, using scaling laws if appropriate. This approach has already been applied to Gl 581c and Gl 581d (von Bloh et al. 2007), as well as to simulations of hypothetical super-Earth planets in various other systems, i.e., 47 UMa and 55 Cnc Franck et al. 2003;von Bloh et al. 2003), as well as systems undergoing red giant branch evolution (von Bloh et al. 2009).
The main question is whether Gl 581g, if existing, offers the principal possibility of life, i.e., whether it lies within the stellar habitable zone (HZ), as already argued by Vogt et al. (2010). Typically, stellar HZs are defined as regions around the central star where the physical conditions are favourable for liquid water to exist at the planet's sur-face for a period of time sufficient for biological evolution to occur. Kasting et al. (1993) calculated the HZ boundaries for the luminosity and effective temperature of the present Sun as R in = 0.82 AU and R out = 1.62 AU. They defined the HZ of an Earth-like planet as the region where liquid water is present at the surface.
According to this definition, the inner boundary of the HZ is determined by the loss of water via photolysis and hydrogen escape. The outer boundary of the HZ is determined by the condensation of CO 2 crystals out of the atmosphere that attenuate the incident sunlight by Mie scattering. The critical CO 2 partial pressure for the onset of this effect is about 5 to 6 bar. However, the cooling effect of CO 2 clouds has been challenged by Forget & Pierrehumbert (1997). CO 2 clouds have the additional effect of reflecting the outgoing thermal radiation back to the surface. The precise inner and outer limits of the climatic habitable zone are still unknown due to the limitations of the existing climate models. Recently, Heller et al. (2011) extended the concept of the HZ by including constraints arising from tidal processes due to the planet's spin orientation and rate. Effects caused by tilt erosion, tidal heating, and tidal equilibrium rotation seem to be especially important for potentially habitable planets (as Gl 581d and Gl 581g) around low-mass stars. For limitations of the planetary habitability of M-type stars see, e.g., Tarter et al. (2007), Guinan &Engle (2009), andSegura et al. (2011).
The luminosity and age of the central star play important roles in the manifestation of habitability. The luminosity of Gl 581 can be obtained by (1) photometry (Bonfils et al. 2005;Udry et al. 2007), and (2) the application of the mass-radius relationship (Ribas 2006) together with the spectroscopically determined stellar effective temperature of T e = 3480 K (Bean et al. 2006). Both methods yield L = 0.013 ± 0.002 L ⊙ . Selsis et al. (2007) estimated the stellar age as at least 7 Gyr based on the non-detection of Gl 581's X-ray flux considering the sensitivity limit of ROSAT (Schmitt et al. 1995;Voges et al. 2000).
In the following, we adopt a definition of the HZ previously used by Franck et al. (2000a,b). Here habitability at all times does not just depend on the parameters of the central star, but also on the properties of the planet. In particular, habitability is linked to the photosynthetic activity of the planet, which in turn depends on the planetary atmospheric CO 2 concentration together with the presence of liquid water, and is thus strongly influenced by the planetary dynamics. We call this definition the photosynthesissustaining habitable zone, pHZ. In principle, this leads to additional spatial and temporal limitations of habitability because the pHZ (defined for a specific type of planet) becomes narrower with time owing to the persistent decrease of the planetary atmospheric CO 2 concentration.
Photosynthesis-sustaining habitable zone (pHZ)
The climatic habitable zone at a given time for a star with luminosity L and effective temperature T e different from the Sun can be calculated following Underwood et al. (2003) based on previous work by Kasting et al. (1993) as with S in (T e ) and S out (T e ) described as second order polynomials.
To assess the habitability of a terrestrial planet, an Earth-system model is applied to calculate the evolution of the temperature and atmospheric CO 2 concentration. On Earth, the carbonate-silicate cycle is the crucial element for a long-term homeostasis under increasing solar luminosity. On geological time-scales, the deeper parts of the Earth are considerable sinks and sources of carbon.
Our numerical model previously applied to Gl 581c and Gl 581d (von Bloh et al. 2007) couples the stellar luminosity L, the silicate-rock weathering rate F wr and the global energy balance to obtain estimates of the partial pressure of atmospheric carbon dioxide P CO 2 , the mean global surface temperature T surf , and the biological productivity Π as a function of time t (Fig. 1). The main point is the persistent balance between the CO 2 sink in the atmosphere-ocean system and the metamorphic (plate tectonic) sources. This is expressed through the dimensionless quantities where f wr (t) ≡ F wr (t)/F wr,0 is the weathering rate, f A (t) ≡ A c (t)/A c,0 is the continental area, and f sr (t) ≡ S(t)/S 0 is the areal spreading rate, which are all normalized by their present values of Earth. Eq.
(2) can be rearranged by introducing the geophysical forcing ratio GFR (Volk 1987) as Here we assume that the weathering rate only depends on the global surface temperature and the atmospheric CO 2 concentration. For the investigation of a super-Earth under external forcing, we adopt a model planet with a prescribed continental area. The fraction of continental area with respect to the total planetary surface f A is varied between 0.1 and 0.9. The connection between the stellar parameters and the planetary climate can be obtained by using a radiation balance equation (Williams 1998) where a denotes the planetary albedo, I R the outgoing infrared flux, and R the distance from the central star. (3) and (4) constitute a set of two coupled equations with two unknowns, T surf and P CO 2 , if the parameterization of the weathering rate, the luminosity, the distance to the central star and the geophysical forcing ratio are specified. Therefore, a numerical solution can be attained in a straightforward manner. The photosynthesis-sustaining HZ around Gl 581 is defined as the spatial domain of all distances R from the central star where the biological productivity is greater than zero, i.e., In our model, biological productivity is considered to be solely a function of the surface temperature and the CO 2 partial pressure in the atmosphere. Our parameterization yields zero productivity for T surf ≤ 0 • C or T surf ≥ 100 • C or P CO2 ≤ 10 −5 bar (Franck et al. 2000a). The inner and outer boundaries of the pHZ do not depend on the detailed parameterization of the biological productivity within the temperature and pressure tolerance window.
Comments on the thermal evolution model
Parameterized convection models are the simplest models for investigating the thermal evolution of terrestrial planets and satellites. They have been successfully applied to the evolution of Mercury, Venus, Earth, Mars, and the Moon (Stevenson et al. 1983;Sleep 2000). Franck & Bounama (1995) studied the thermal and volatile history of Earth and Venus in the framework of comparative planetology. The internal structure of massive terrestrial planets with one to ten Earth masses has been investigated by Valencia et al. (2006) to obtain scaling laws for total radius, mantle thickness, core size, and average density as a function of mass. Similar scaling laws were found for different compositions. We will use these scaling laws for the mass-dependent properties of super-Earths and also the mass-independent material properties given by Franck & Bounama (1995). The thermal history and future of a super-Earth has to be determined to calculate the spreading rate for solving key Eq. (2). A parameterized model of whole mantle convection including the volatile exchange between the mantle and surface reservoirs (Franck & Bounama 1995;Franck 1998) is applied. The key equations used in our present study are in accord with our previous work focused on Gl 581c and Gl 581d; see von Bloh et al. (2007), for details. A key element is the computation of the areal spreading rate S; note that S is a function of the average mantle temperature T m , the surface temperature T surf , the heat flow from the mantle q m , and the area of ocean basins A 0 (Turcotte & Schubert 2002). It is given as where κ is the thermal diffusivity and k the thermal conductivity. To calculate the spreading rate, the thermal evolution of the mantle has be to computed: (7) where ρ is the density, c is the specific heat at constant pressure, E is the energy production rate by decay of radiogenic heat sources in the mantle per unit volume, and R m and R c are the outer and inner radii of the mantle, respectively. To calculate the thermal evolution for a planet with several Earth masses, i.e., 3.1 and 4.3 M ⊕ as pursued for Gl 581g in our present study, the planetary parameters have to be adjusted. Thus we assume where R p is the planetary radius, see Valencia et al. (2006). The total radius, mantle thickness, core size, and average density are all functions of mass, with the subscript ⊕ denoting Earth values. The exponent of 0.27 has been obtained for super-Earths. The values of R p , R m , R c , as well as the other planetary properties are scaled accordingly. It means that R p , R m and R c increase by a factor of 1.36 for M = 3.1M ⊕ and 1.48 for M = 4.3M ⊕ . Table 1 gives a summary of the size parameters for the models of the planets with 3.1 and 4.3 M ⊕ ; see the study by von Bloh et al. (2007) for additional information. The values for an Earth-size planet are included for comparison. The onset of plate tectonics on massive terrestrial planets is a topic of controversy. While O'Neill & Lenardic (2007) stated that there might be in an episodic or stagnant lid regime, Valencia & O'Connell (2009) proposed that a more massive planet is likely to convect in a plate tectonic regime similar to Earth. Thus, the more massive the planet is, the higher the Rayleigh number that controls convection, the thinner the top boundary layer (lithosphere), and the higher the convective velocities. In the framework of our model, a plate-tectonic-driven carbon cycle is considered necessary for carbon-based life. This approach follows the previous work by von Bloh et al. (2007), who gave a detailed discussion of the equations and parameters used in their study for the super-Earths with 5 and 8 M ⊕ , identified as Gl 581c and Gl 581d, respectively.
Results and discussion
The photosynthesis-sustaining habitable zone is calculated for super-Earth planets with 3.1 and 4.3 M ⊙ and the results are depicted in Fig. 2. The maximum life span of a biosphere is given at the point in time when the pHZ vanishes. This maximum life span strongly depends on the relative continental area r and increases with decreasing r. Therefore, "water worlds" are favored in the facilitation of habitability as previously obtained in models of fictitious super-Earth planets for 47 UMa and 55 Cnc von Bloh et al. 2003). In this context, water worlds are planets of non-vanishing continental area mostly covered by oceans. The climate of a planet fully covered by oceans is not stabilized by the carbonate-silicate cycle. The maximum life span for a water world with r = 0.1 and a planetary mass of M = 3.1M ⊕ is 15.4 Gyr, whereas it is 16.3 Gyr for M = 4.3M ⊕ . Both times are considerably longer than the estimated age limit of Gl 581, which is 7 Gyr (Selsis et al. 2007). This maximum life span can be realized for a super-Earth at a distance from the central star of about 0.14 ± 0.015 AU. This uncertainty is reflective of the uncertainty in the luminosity estimate of Gl 581, noting however that our models are also subject to various systematic uncertainties (see Sect. 2). A full assessment of those uncertainties, which are expected to be larger than the impact of the uncertainty in the stellar luminosity, can best be obtained by a comparison with future alternative models. It is noteworthy that the orbital position of Gl 581g at 0.146 AU, if existing, is largely consistent with this ideal position. Thus, we can conclude that Gl 581g has an almost perfect Goldilock position in the star-planet system of Gl 581, and it is the best candidate for extra-terrestrial habitability so far. Because of the Goldilock position, located well within the HZ, clouds play no major role for our results. Any shift (within limits) of the inner or outer boundary of the HZ will not affect the maximum life span obtained by our model. The optimum position is not only the geometric mean of the pHZ, but it is also the position where the maximum life span of the biosphere is realized.
But the pHZ also strongly depends on the planetary age. Figure 3 depicts the life span for a super-Earth with 3.1 and 4.3 M ⊕ at 0.146 AU as a function of the relative continental area r. For a relative continental area larger than 0.6, the realized life span is shorter than 7 Gyr, which is the estimated lower limit for the stellar age of Gl 581 star-planet system. In this case, no habitable conditions on Gl 581g would exist in the framework of the adopted geodynamic model. If we place an Earth twin (r ≃ 0.29) at the orbital position of Gl 581g, habitable conditions would cease just at an age of 7 Gyr. In conclusion, we have to await future missions to identify the pertinent geodynamical features of Gl 581g (in case it does exist) and to search for biosignatures in its atmosphere to gain insight into whether or not Gl 581g harbors life. | 2011-02-22T22:20:07.000Z | 2011-02-18T00:00:00.000 | {
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13972783 | pes2o/s2orc | v3-fos-license | Multiscale-structuring of polyvinylidene fl uoride for energy harvesting: the impact of molecular-, micro- and macro-structure of Materials Chemistry A REVIEW
Energy harvesting exploits ambient sources of energy such as mechanical loads, vibrations, human motion, waste heat, light or chemical sources and converts them into useful electrical energy. The applications for energy harvesting include low power electronics or wireless sensing at relatively lower power levels (nW to mW) with an aim to reduce a reliance on batteries or electrical power via cables and realise fully autonomous and self-powered systems. This review focuses on fl exible energy harvesting system based on polyvinylidene fl uoride based polymers, with an emphasis on manipulating and optimising the properties and performance of the polymeric materials and related nanocomposites through structuring the material at multiple scales. Ferroelectric properties are described and the potential of using the polarisation of the materials for vibration and thermal harvesting using piezo- and pyro-electric e ff ects are explained. Approaches to tailor the ferroelectric, piezoelectric and pyroelectric properties of polymer materials are explored in detail; these include the in fl uence of polymer processing conditions, heat treatment, nanocon fi nement, blending, forming nanocomposites and electrospinning. Finally, examples of fl exible harvesting devices that utilise the optimised ferroelectric polymer or nanocomposite systems are described and potential applications and future directions of research explored.
Introduction
Energy harvesting is a topic of intense interest where ambient sources of energy are harvested and converted into useful electrical energy. The concept of harvesting energy covers applications that produce relatively small levels of power (nW to mW) from mechanical loads, vibrations, human motion, waste heat, light or chemical sources. The power generated by the harvesting material or device is used for applications such as low power electronics, lighting or wireless sensor systems. The ability to use energy harvesting to produce autonomous selfpowered systems is of interest to reduce reliance on power cables or batteries that require regular replacement or recharging; there are also environmental benets from reduced battery usage.
Piezoelectric and pyroelectric materials are particularly attractive for a variety of energy harvesting applications. This includes the potential to convert mechanical vibrations into electrical energy via the direct piezoelectric effect and the conversion of thermal uctuations into electrical energy via the pyroelectric effect. Since both properties are oen present simultaneously, it provides a potential route to harvest energy from multiple sources so that the design of hybrid systems is possible.
A number of excellent reviews have been published in the area of piezoelectric and pyroelectric energy harvesting, which concentrate on nano-scale materials and devices, including 'nanogenerators', [1][2][3][4][5][6][7][8][9][10] and surveys of the various potential devices. [11][12][13][14][15][16] Li et al. recently presented a review on ferroelectric polymers for energy, including storage applications 17 and Fan et al. reviewed exible nanogenerators for harvesting and selfpowered electronics. 18 Prateek et al. 19 have overviewed recent progress on ferroelectric polymer based nanocomposite for energy storage and capacitor applications. The emphasis of this review is to describe mechanically exible systems based on polyvinylidene uoride (PVDF) polymers for energy harvesting, with an emphasis on manipulating the properties and energy harvesting performance of ferroelectric polymers and polymer nanocomposites through structuring the materials at multiple scales. This includes effort to tailor the ferroelectric, piezoelectric and pyroelectric properties of polymer materials by manipulating the polymer processing conditions, heat treatment, nano-connement, blending, forming nanocomposites, electrospinning and creating porous structures. PVDF is chosen in this review as it the most commonly used piezoelectric polymer due to its high piezoelectric coefficient compared with other bulk polymers. A comparison with other piezoelectric materials will be undertaken later in Table 1 and a detailed comparison of the range of piezoelectric polymers has been undertaken by Ramadan et al. 20 Before discussing the properties of ferroelectric PVDF-based polymers it is rst of interest to introduce the mechanisms of harvesting via the piezoelectric and pyroelectric effects so that the important properties for energy harvesting can be identied.
Piezoelectric and ferroelectric materials
Piezoelectric materials undergo a change in electrical polarisation when a mechanical stress is applied and this is termed the direct piezoelectric effect. This effect can create an electrical current in an external circuit and can therefore be exploited as an electromechanical generator to harvest energy. Piezoelectric materials also undergo a mechanical strain when subjected to an electric eld, this is termed the converse piezoelectric effect and is more relevant to actuator, acoustic emitter and vibration damping applications and will not discussed here.
The phenomenon of piezoelectricity is best introduced by considering a crystalline solid and its distribution of ions within an individual unit cell. The most common case is when the centres of the positive and negative charges are noncentrosymmetric within the unit cell planes when no pressure is applied; this is the case in ceramics such as barium titanate (BaTiO 3 ), lead zirconate titanate (PZT), aluminium nitride (AlN) and zinc oxide (ZnO). Of the 32 crystallographic classes that depend on the geometry and symmetry of the unit cell, 21 are non-centrosymmetric and all but one (due to other symmetry elements) exhibit piezoelectricity. It is the lack of symmetry in the distributions of ions in these crystalline materials that lead to the presence of an electrical dipole that leads to the piezoelectric response. Due to the lack of symmetry, piezoelectric materials for energy harvesting possess a well-dened polar axis and the application of stress relative to this polar axis is of importance for energy harvesting applications; the polar axis is oen termed the 3-direction or z-direction.
Ferroelectrics are a subgroup of piezoelectric materials, and are characterised by a spontaneous polarisation in the unstrained state and the capability to re-orientate the polarisation direction via an applied electric eld. The BaTiO 3 and the PZT family of ceramic materials are commonly used ferroelectric ceramics due to their good piezoelectric properties. The subject of this review is ferroelectric polymers, such as polyvinylidene uoride (PVDF) and its copolymers. Example copolymers include polyvinylidene-diuoride triuoro-ethane, P(VDF-co-TrFE), and we will see later that these are crystalline polymers with aligned molecular chains where the polarity is due to the alignment of polarised covalent bonds. The temperature limits of a ferroelectric are determined by the Curie temperature (T c ) and when BaTiO 3 is heated above T c its crystal structure is cubic and since the unit cell is symmetrical there is no polarisation and the material in no longer ferroelectric. Below the T c the unit cell is tetragonal and non-centrosymmetric, thereby leading to ferroelectric properties. A similar behaviour is observed in PVDF based materials where the ferroelectric b-phase converts to the paraelectric a-phase when the material is heated above T c .
While a ferroelectric contains many individual unit cells with a corresponding electrical dipole, there are regions where the unit cells have equal polarisation directions, these are known as domains. When a ferroelectric is cooled below the T c there is no net polarisation as the domains are effectively randomly distributed through the volume of the material. For the material to exhibit piezoelectricity the randomly orientated electrical dipoles are aligned in a common direction to achieve a net polarisation in a process called 'poling'. The process of electrical alignment, or 'poling', is essential in converting an inactive ferroelectric polymer into a useful electromechanically active material. Poling involves the application of an electric 57) eld, oen at elevated temperatures, to orientate the polar axis of the domains to those directions allowed by the crystal symmetry, which are nearest to that of the applied electric eld.
To further facilitate the poling process ferroelectric polymers oen require mechanical stretching to enhance the alignment of the molecular chains and these factors will be discussed later in the review. Fig. 1 shows a simple schematic of the poling process and the material initially consists of randomly orientated domains (Fig. 1a). The material is then heated to an elevated temperature below T c , and an electric eld is applied to orientate the domains (Fig. 1b). The material is then cooled to ambient temperature while the electric eld remains applied. On removal of the electric eld at ambient temperature, although some domains may change conguration (Fig. 1c), the material retains a net polarisation producing a poled ferroelectric with piezoelectric behaviour. Once poled, the material has a polar axis (the z-or 3-direction), which for the case in Fig. 1a-c is through the thickness of the material. The material is now piezoelectric and will exhibit the direct and converse piezoelectric effects.
A polarisation-electric eld (P-E f ) loop of PVDF is shown in Fig. 1d. At point 1 the material has no net polarisation, as the dipoles are randomly orientated (as in Fig. 1a). However, as the electric eld is applied the dipoles align with the applied electric eld to produce a polarisation at point 2 (as in Fig. 1b). Once the electric eld is removed, the PVDF has a remanent polarisation (P r ), where the material is similar to Fig. 1c and the material is piezoelectric. Clearly a high P r is of interest to achieve a high piezoelectric activity for harvesting applications. If an electric eld is applied in the opposite direction the polarisation can be returned to zero (point 4) and the polarisation direction can be reversed; which is dened as the coercive eld (E c ).
Direct piezoelectric effect for energy harvesting
The direct piezoelectric effect, where a force leads to the production of an electrical charge, can be used for energy harvesting. The presence of a polarisation with aligned dipoles and domains leads to the presence of a charge at each surface of the material and free charges, such as ions or electrons, are attracted to the charged surfaces of the material. This is shown schematically in Fig. 2a where the polarisation of the material is observed along with its bound surface charge. No current will ow under this stress-free condition if an electrical load it connected across the surfaces. The origin of the direct piezoelectric effect stems from the behaviour of the surface charge as the material is subjected to a stress that changes the polarisation level. If the application of a compressive stress leads to a decrease in the polarisation of the material, as in Fig. 2b, the free charges at the surface generate a current across an electrical load applied to the material. In Fig. 2c the application of a tensile load in the opposite direction increases the polarisation level and current ows in the opposite direction to balance the surface charge. If an alternating stress in applied to the piezoelectric the piezoelectric will generate an AC current through the load impedance.
The charge (Q) generated across the opposite faces of a piezoelectric material of area (A) when a mechanical stress (Ds) is applied between the faces is given by eqn (1). 22 where d 33 is the piezoelectric coefficient (C N À1 ); the 33-mode corresponds to the application of stress in the polarisation direction, which is the case in Fig. 2. Other modes can be utilised to harvest energy, for example in the 31-mode the applied stress is normal to the polarisation direction and d 15 is a shear mode. It is possible to determine typical voltages and current generated under an applied stress by considering two extremes of load impedance in Fig. 2. Under open circuit conditions, where the load impedance is innite, we can use the relationship Q ¼ CV, where C (¼A Â 3 T 33 /h) is the material capacitance to determine the voltage (V) from eqn (2).
where h is the thickness and 3 T 33 is the permittivity at constant stress in the polarisation direction. Since the energy stored in a capacitor in 1/2CV 2 , the energy (E) as a result of an applied stress is given by eqn (3). Therefore, for a given area and thickness the energy from an applied stress can be maximised by selecting materials with a high d 33 ; this is termed a harvesting gure of merit (FoM) to assess the performance of a material for vibration harvesting off-resonance. Under short circuit conditions, where the load impedance is zero, a current (I) will be generated and since (1) we arrive at eqn (4).
Measurements at both open circuit and closed circuit are commonly made in energy harvesting applications, although it should be noted at these conditions there is effectively no power since at open circuit conditions there is no current and at closed circuit conditions the potential difference is zero.
Pyroelectric effect for energy harvesting
While the piezoelectric effect is the generation of charge with applied stress, the pyroelectric effect is the generation of charge with a temperature change. As with piezoelectric materials, pyroelectrics are polar materials and exhibit a spontaneous polarization in the absence of an applied electric eld 23 and in ferroelectric polymers the polarisation is a consequence of the alignment of polarised covalent bonds. 24 As described in Section 1.2, the polarisation leads to the presence of bound charge on each surface of the material, as in Fig. 3a, and the origin of pyroelectric behaviour stems from the change in polarisation level with material temperature. 23 If a pyroelectric is heated (dT/dt > 0), as in Fig. 3b, there is a decrease in its polarisation as dipoles within the material lose their orientation due to thermal vibrations. This fall in the polarisation leads to a decrease in the number of free charges bound to the material surface; 23 see Fig. 3c. If the material is under an open circuit condition the free charges remain at the electrode surface and an electric potential is generated across the material. 25 If the material is under short circuit conditions an electric current ows between the two polar surfaces of the material. If the pyroelectric is then cooled (dT/dt < 0), as in Fig. 3b, the dipoles regain their orientation leading to an increase in the level of spontaneous polarization, thus reversing the electric current ow under short circuit conditions as free charges are now attracted to the polar surfaces; see Fig. 3d.
Eqn (5) denes the relationship between the developed pyroelectric charge (Q) due to a change in temperature (DT) for a material of surface area (A) with a pyroelectric coefficient (p). 26 Since current is dQ/dt, eqn (6) provides the short circuit pyroelectric current (i p ) as a function of the rate of temperature change (dT/dt) 27 with electrodes orientated normal to the polar direction.
The pyroelectric coefficient of an unclamped material, under a constant stress and electric eld, is dened by eqn (7), where P s is spontaneous polarisation 28 and subscripts s and E correspond to conditions of constant stress and electric eld respectively. We have seen for vibration harvesting there is a need to dene the direction of applied load relative to the poling direction (e.g. 33-and 31-mode) and while the pyroelectric coefficient is a vector quantity, the electrodes that collect the charges are oen normal to the polar direction and so the measured quantity is oen treated as a scalar. 29 Under opencircuit conditions the charge created due to a temperature change, leads to a potential difference across the material, given by eqn (8).
Under open circuit conditions the material behaves as a capacitor and the energy stored in the pyroelectric is 1/2CV 2 , such that the energy (E) generated from a change in temperature is given by eqn (9).
Therefore, for a given area and thickness the energy for a temperature change can be maximised by maximising selecting a materials with a high p 2 3 T
33
; which is a gure of merit to asses the performance of a material for pyroelectric harvesting. This gure of merit has similarities with the vibration gure of merit d 33 2 3 T 33 which is not surprising given the similarity in the charge generation for both mechanisms result from a change in polarisation of the material as a result of stress, d ij ¼ dP s /ds (C N À1 or C m À2 /N m À2 ), and temperature, p ¼ dP s /dT (C m À2 K À1 ). Since there is a requirement for a pyroelectric material to be polar and exhibit a level of polarisation all pyroelectric materials are piezoelectric (pyroelectrics are a sub-class of piezoelectric materials so that all pyroelectrics are also piezoelectric). In ferroelectric pyroelectric materials, such as PVDF, the orientation, and sign, of the spontaneous polarisation can be switched by reversing the direction of the applied electric eld. These ferroelectrics are a sub-class of pyroelectric materials so that all ferroelectrics are both pyroelectric and piezoelectric.
Clearly for energy harvesting applications the gures of merit indicate that there is a need for high piezoelectric activity to maximise the piezoelectric coefficients (such as d 33 and d 31 ) and high pyroelectric coefficients (p); this also relates to a need for a high remnant polarisation (P r ) aer the poling process. Since the origins of the piezoelectric and pyroelectric effect originate from an electrical dipole from the polymer molecule there is a need to maximise the fraction of the non-symmetrical phase of the polymer and ensure it can be easily poled. The gures or merit also indicate a low relative permittivity is benecial for harvesting, although a large electrical dipole or ease of polarisation oen leads to a high permittivity so that the optimum choice of material based on a combination of d 33 , d 31 , p and 3 T 33 can be complex. Other factors include the Curie temperature (T c ) since it indicates the temperature at which the polymer transforms from a ferroelectric to paraelectric phase and indicates the temperature at which piezoelectric/pyroelectric properties are lost. The ferroelectric phase of polymers such as PVDF and its copolymers will now be discussed in more detail.
Piezo-and pyro-electric polymers
We have seen that ferroelectric and piezoelectric materials can be ceramic or polymeric. Electroactive polymers have obvious advantages over ceramics in specic applications in terms of their ease of processing at low-temperatures, low density, low stiffness, exibility and mechanical robustness, such as toughness and high strains to failure. There are also benets in terms of biocompatibility for implantable harvesters and sensors. 30 Polymer based piezoelectrics have found diverse applications in smart and multifunctional systems which include transducers, sensors, actuators, energy harvesting and storage devices, in the form of bres, foams, thin lms, textiles and coatings. Ferroelectric polymers that contain net molecular dipole moments in their macromolecular structure are of interest for energy harvesting and storage due to their high levels of polarisation. The molecular dipoles in these materials can be spontaneously polarised and oriented by the application of an electric eld, temperature variation, mechanical stretching, and via interactions with nanoparticles; 31 thereby leading to piezo-and pyro-electric response of the polymers. Polarised and electroactive polymers of interest include vinylidene uoride (VDF)-based uoropolymers, odd-numbered nylon, poly-L-lactide, polyurethane, and liquid crystal elastomers. The electroactive performance is highly dependent on their macromolecular structure which includes aspects of their crystalline structure, chain conformation, dipole orientation in crystalline regions towards the applied poling eld, 31,32 processing conditions and post-treatment methods. Compared to ferroelectric ceramics, electroactive polymers tend to have a lower permittivity and lower piezoelectric coef-cients. Table 1 shows typical properties of electroactive polymers along with common piezoelectric ceramics. Due to their relatively low piezoelectric activity, signicant effort has been undertaken to enhance the piezoelectric coefficients of polymers and tailor the permittivity, and the approaches used to improve the electrical properties include modifying the molecular structure, forming composites, controlling processing conditions and post-treatment methods such as mechanical stretching and electrical poling. In this review we will discuss the latest research progress and strategies employed to enhance the piezo-and pyro-electric properties of electroactive polymers and composites, in particular PVDF and its copolymers due to their high piezoelectric coefficient and ease of fabrication, with a particular emphasis on energy harvesting applications. structure and variety of crystalline forms. Table 1 indicates PVDF is a polymer with high piezoelectric coefficients compared to other polymers and the origins of the negative piezoelectric coefficients of PVDF in Table 1 has recently been reported by Katsouras et al. 58 The PVDF homopolymer contains 59.4 wt% uorine and 3 wt% hydrogen. 31 The presence of uorine atoms with a large van der Waals radius (1.35Å, versus hydrogen 1.2Å) and electronegativity in the polymer chain [-CH 2 -CF 2 -] induces a dipole moment perpendicular to the chain in each monomer unit. 32,59 PVDF has approximately 50% crystallinity, and exhibits ve different polymorphs: a (phase II), b (phase I), g (phase III), d and 3, which are related to the molecular chain conformations. Fig. 4a shows the a, b and g phases. 42 The a-crystal phase is hexagonal, with aligned polymer chains anti-parallel to each other, in the conformation of trans-gauche-trans-gauche 0 (TGTG 0 ). 42 The polar b-crystal is orthorhombic and has an alltrans planar zigzag conformation with their dipoles parallel to the b-axis, and contribute to the highest dipolar moment per unit cell (8 Â 10 À30 C m). 60 The phase transition from the ferroelectric b-phase to the paraelectric a-phase is dened as the Curie transition, which is a process that is highly dependent on the polymer chain structure, processing condition and posttreatment. Achieving a higher b-phase fraction in PVDF leads to higher piezo-, pyro-and ferroelectric properties. 31,32,34 The polar gand d-phase have the conformation of TTTTGTTTG 0 and TGTG 0 , respectively, which are also responsible for the piezo-and pyro-electric properties of PVDF, together with the b-phase. 31 2.1.1 Characterisation of ferroelectric polymers. The polymorphs of ferroelectric polymers are generally identied and quantied by using combined Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC) techniques. FTIR is an important approach to evaluate the polar b-phase content, a to b transition, as well as the dipolar orientation in the material. With regards to the FTIR spectra of PVDF, the vibrational bands at 530 cm À1 (CF 2 bending), 615 cm À1 and 765 cm À1 (CF 2 bending and skeletal bending) and 795 cm À1 (CH 2 rocking) refer to the aphase; the vibrational bands at 510 cm À1 (CF 2 bending) and 840 cm À1 (CH 2 rocking) correspond to the ferroelectric b-phase. The fraction of b-phase, F (b) , in the complete crystalline phase can be determined with eqn (10) 62 in which X b and X a are the crystalline mass fractions of aand bphase and A a and A b correspond to the absorbance intensities at 764 and 841 cm À1 , respectively. The K 841 (7.7 Â 10 4 cm 2 mol À1 ) and K 764 (6.1 Â 10 4 cm 2 mol À1 ) parameters are the absorption coefficients at the respective wavenumbers. 32 The polar g-phase has a similar chain conformation as the b-phase, and share similar characteristic peaks in the FTIR spectrum. The polymorph can be also identied by XRD. The characteristic peaks at 2q ¼ 17.6 , 18.3 , 19.9 , and 26.5 correspond to the (100), (020), (110), and (021) reections of the a-phase of PVDF. The (200) and (110) reections of the b-phase and (021) reection of the g-phase overlap at 2q ¼ 20.4 . 32,59 Differential scanning calorimetry (DSC) is oen used to study the Curie transition and crystallisation behaviour of polymers during a change from the ferroelectric b-phase to the paraelectric a-phase. Measurements are generally conducted in the temperature range of 0 to 220 C for PVDF and its copolymers, to understand the relationship of heat ow and temperature during non-isothermal and isothermal crystallisation processes. A value of 104.6 J g À1 was used as the heat of fusion of the perfect crystalline a-phase of PVDF. 63 The efficiency of nucleation agents can be evaluated using eqn (11); 64 where T c-nucl , T c1 , T c2 are the crystallization temperatures of the nucleated, non-nucleated and self-nucleated polymer, respectively. The melting temperature and crystalline temperature of polymers are dependent on the molecular structure, molecular weight and any annealing process. The surface morphology, chain alignment and dipole orientation, ferroelectric properties have been characterised by FTIR-grazing incident reection absorption spectroscopy, grazing incident wide angle X-ray diffraction, 65 atomic force microscopy, small-angle neutron scattering, 66 dynamic contact electrostatic force microscopy, and polarization-electric eld hysteresis measurements. 67 Measurements of the ferroelectric and piezoelectric properties can be undertaken by polarisation-electric eld measurements, piezo-response force microscopy and second harmonic generation microscopy.
2.1.2 Formation of ferroelectric b-phase. A variety of methods have been developed to increase the fraction of the ferroelectric b-polymorph in PVDF, including co-polymerisation with a second monomer, blending with other polymers, forming composites with nucleating agents or nanollers, tailoring the processing conditions, mechanical stretching and electrical poling. The formation of the non-polar a-phase of PVDF is kinetically more favourable which can readily form by crystallization from the melt at moderate or high levels of supercooling 68 (<160 C), or from solution-crystallisation of a xylene/ acetone mixture, monochlorobenzene, or dimethylformamide (DMF) solutions. 62 While the polar b-phase is the most thermodynamically polymorph, it can only be formed under special conditions. These include (i) melt crystallization at high pressure 65 or very high cooling rates, 68,69 (ii) solution-casting from highly polar solutions, such as hexamethyl phosphoramide, 70 (iii) vapour deposition of oligomeric PVDF 71,72 or (iv) by blending with surface charged nanoparticles or nucleating agents. The aphase can transform to the b-phase by mechanical-stretching and electrically poling at electric elds as high as 100 kV mm À1 under elevated temperature of 80-165 C. 31 From rst-principle simulations and a generalised solid-state nudged elastic band method, 73 it was suggested that the electric eld transforms the a-phase to the polar d-phase, and then to the b-phase. This is shown in Fig. 4b where the a-phase forms from the melt and progressively transforms to the b-phase at high electric elds. The inuence of cold drawing and annealing at high pressure to form the b-phase is also shown in Fig. 4b. 61 The crystalline structure of PVDF has been studied during a micro-injection process and in the presence of polar additives. 74 In contrast to crystallisation in the static state, the application of high stress during micro-injection promotes the formation of b-phase. The presence of polar additives also aids in the stabilization of the metastable TT conformation (as shown in Fig. 4b) via an interaction between the polar groups and the pCF 2 groups of PVDF, which benets the formation of b-PVDF. As a result, PVDF micro-components with rich b-phases have been injection-moulded under the combined effects of an externally applied force and polar group interaction.
The a to b transformation is highly dependent on the stretching rate and temperature. By stretching PVDF blown lms biaxially or uniaxially, 75 a high 86.5% content of highly oriented b-phase was achieved during drawing at 87 C, with a drawing rate of 50 mm min À1 and a stretch ratio of 6.5. At this stretch ratio, a high d 33 piezoelectric coefficient of 33 pC N À1 was obtained. Other studies have shown that the b-phase is more favourably formed in the temperature range of 70-100 C and at a stretch ratio of 3-5. 76 Higher stretching temperatures reduce the efficiency of the phase conversion to b-phase and the conversion into b-phase only takes place for stretch ratios above 5. It has also been shown that annealing of the PVDF thin lms at 90 C transforms the g-phase to the b-phase and this is re-ected in a decrease of the relative permittivity. 77 Recently, nanofabrication technologies such as electrospinning, nanoimprint lithography 78 and the horizontal Langmuir-Schaefer technique 79 have attracted attention to produce non-volatile memory and exible piezoelectric sensors. In particular, electrospinning can produce a high b-phase content in PVDF nanobers via a one-step process that avoids additional processing stages such as mechanical stretching or electrical poling; this process will be described later in Section 2.2.8. In the following sections, the latest progress in understanding the effects of co-polymerisation, processing condition, post-treatment, addition of nanoparticles and electrospinning on the piezo-and pyro-electric properties of PVDF and its copolymers are discussed. Harvesting devices exploiting these optimised properties are then described.
2.2 Approaches to enhancing b-phase formation 2.2.1 Copolymerisation and phase transition. Copolymerisation is an effective method to tune the polymorph structure and phase transition behavior of PVDF. The comonomer type and composition ratio determine the crystalline structure of the polymer and degree of crystallinity which directly affect the ferro-, piezo-and pyro-electric properties. A variety of VDF-copolymers and terpolymers have been synthesized by incorporation of triuoroethylene (TrFE), chlorotri-uoroethylene (CTFE) or hexauoropropylene (HEP) comonomer. The molecular structures of these commonly studied monomers and the copolymers are shown in Fig. 5 and typical properties have been shown in Table 1 for comparison.
The introduction of a bulky co-monomer to PVDF chains generally facilitates the formation of ferroelectric b-phase due to a steric hindrance effect. 80 For example, for P(VDF-co-TrFE) in Fig. 5a the introduction of a third uoride atom in the TrFE [-CHF-CF 2 -] co-monomer unit forces the polymer chains to align in an extended planar zigzag all-trans conformation below T c when the TrFE content is over 11 mol%. 81 When the TrFE is above 20 mol%, the formation of b-phase is independent of the processing conditions and electric poling. 82 For P(VDF-co-HFP), shown in Fig. 5b, the presence of bulky -CF 3 groups in the PVDF chains provides more space to allow dipoles to re-orient. With a HFP content of 5 mol%, a high value of remnant polarization (P r $ 80 mC m À2 ) was achieved for solvent cast lms, resulting in a high d 31 piezoelectric coefficient of 30 pC N À1 . 83 In the case of the P(VDF-co-CTFE) copolymer, Fig. 5c, a CTFE content lower than 16 mol% (ref. 42) led to a d 33 piezoelectric coefficient as high as 140 pC N À1 . 43 Therefore, the advantages of PVDF copolymers over PVDF is that the b-phase is always present regardless of processing methods, which can be attributed to the steric hindrance of the co-monomer that expands the interchain distance and reduces the activation energy for the a to b phase transition. 32,84 Additional annealing, mechanical stretching or electrical poling processes can be used to increase the degree of crystallinity and further align the CF 2 dipoles, which leads to higher piezo-and pyro-electric properties than PVDF homopolymers. By introducing a chloro-containing third monomer termonomer, such as chlorotriuoroethylene (CTFE), to P(VDF-co-TrFE) copolymers, as in Fig. 5d, some of the ferroelectric b-phase transforms to the g-phase when the termonomer units reach 7 mol%. 85 As a result, the T c of the mixed ferroelectric phase polymer was reduced to almost ambient temperature and exhibits a high relative permittivity (3 0 > 70), a slim polarization hysteresis and ferroelectric relaxor behaviour.
For ter-polymers, such as poly(VDF-TrFE-TFP), the termonomer 3,3,3-triuoropropene (TFP) with a bulky side group leads to -CF 3 behaving as crystalline defects in the polymer chains and this results in small crystals and a small degree of crystallinity. This is reected by a T c that reduces from 72 C for a conventional P(VDF-co-TrFE) copolymer to 65 C for the terpolymer. 86 The TFP is mainly an amorphous phase and the bulky -CF 3 groups hinders the mobility of the chain segments, leading to an increase of the glass transition temperature (T g ) from approximately À20 C for a standard copolymer to 0 C for a terpolymer with 9 mol% of TFP. The effect of the ter-monomer on the polarization behaviour of copolymer thin lms (20 mm) were compared for P(VDF-co-TrFE) (65/35), poly(VDF-TrFE-CTFE) (61/26/13) and poly(VDF-TrFE-TFP) (62/32/6). As shown in Fig. 6, the poly(VDF-TrFE-CTFE) exhibited a typical relaxor ferroelectric behaviour with a slim hysteresis loop and low coercive eld (E c ¼ 13 MV m À1 ), remnant polarization (P r ¼ 10 mC m À2 ) and saturation polarization (P sat ¼ 58 mC m À2 ). Both P(VDF-co-TrFE) and poly(VDF-TrFE-TFP) exhibited wide ferroelectric hysteresis loops with a high coercive eld, E c ¼ 63 MV m À1 , indicating that both polymers have a similar polarisation response. The poly(VDF-TrFE-TFP) has a lower P r ¼ 33 mC m À2 and saturation polarisation, P sat ¼ 49 mC m À2 , as compared to P(VDF-co-TrFE) copolymer, which may be due to the lower crystallinity of the terpolymer and the lower polarizability of the -CF 3 group of the TFP compared to the CFE and CTFE units. 86 2.2.2 Processing conditions and inuence on Curie temperature. As shown in Fig. 4b, the paraelectric a-phase is generally formed in PVDF on cooling from the melt or from solution-cast lms. A mixture of a-, b-, or g-phases is generally observed in PVDF copolymers when crystallised from the melt or from solutions under a range of conditions. The conversion of the ferroelectric b-phase to the paraelectric a-phase takes place thermally when the material is heated above the T c , which denes the upper use temperature for piezoelectric and pyroelectric applications such as energy harvesting. 31 The T c of b-PVDF is typically $170 C (ref. 87) and the introduction of a co-monomer to the polymer chains can restrict the degree of crystal growth and form nanosized crystalline domains, 61,85 which increases the inter-chain distance and dipolar mobility, and decreases the T c . For example, a low T c of 105 C was measured for a copolymer of 70/30 composition ratio. 88 A previous study 89 demonstrated that T c is 70-80 C for a 65/ 35 mol% P(VDF-co-TrFE) copolymer, which was observed as a decrease in the intensity of the XRD diffraction peak at 2q ¼ 19.5 (b-phase), and the emergence of a second peak at 2q ¼ 18.2 (a-phase) as the temperature increased above 80 C. The all-trans peak vanished at 100 C (lower than the melting temperature, T m $ 153 C). In the temperature between T c and T m , the polymer chains move more freely to reorganise into higher degree of crystallinity. In comparison, a copolymer of 73/ 27 mol% had a T c of 100-110 C and the all-trans ferroelectric peak disappeared on heating to 140 C, while T m was 150 C. For a 78/22 mol% copolymer, the T c was increased further to 130-145 C and T m was estimated to be 149 C. 89,90 For a 81/19 mol% copolymer, the T c was $120 C, and the T m was near 150 C. 81 During the manufacturing process, polymers suffer from tension, shearing and compression during melt-extrusion, injection moulding and compression moulding; and tension and electrical poling during the electrospinning process. In addition to mechanical stretching or electrical poling, the polar b-phase of PVDF can also be induced by application of a shearing history (shear rate, shear strain and shear temperature). 91 A recent study found an increase in b-phase content from 38 to 84% for PVDF 120-150 mm thick lms when sheared at 220 C with an applied shear rate of 10 s À1 for 10 s, followed by isothermal crystallization at 155 C. The samples sheared at a higher temperature generally had a higher nuclei density, smaller spherulites and a higher b-phase fraction, as shown in Fig. 7. A large deviation between the shearing temperature and crystallization temperature was shown to facilitate the formation of b-phase.
2.2.3 Inuence of annealing conditions on phase transition. The applied annealing treatment affects two important phase transition temperatures, namely the Curie temperature and melting temperature. The T c of PVDF copolymers relies on the chemical composition of the copolymers, and is also determined by the polymer annealing conditions. Copolymers with 50-80% VDF show a T c in the range of 70-140 C, 92 which is absent in pure PVDF.
Generally, a higher annealing or crystallisation temperature and/or longer annealing time above T c yields lamellar thickening and improved crystalline packing, lower defect density and higher crystallinity of the paraelectric a-phase. These factors result in an increase in T m and decrease T c . 67,92 When annealing the polymer below T c in the b-phase state, an increase of the T c is expected. 93 Solution-cast P(VDF-co-TrFE) (72/28) thin lms annealed at 120 C for 3 h (above T c , but below T m ) had a preferential chain orientation that was aligned parallel to the substrate surface with a high degree of crystallinity, and high dipole alignment upon electrical poling. These factors lead to a large polarization in 100 nm thin lms, P r ¼ 7.8 mC cm À2 and an E c ¼ 0.75 MV cm À1 . 67 Therefore, to achieve high piezoelectric coefficients, it is essential to control the annealing procedures through the T c . 92 As an example of the inuence of such processing conditions, for a solution-cast P(VDF-co-TrFE) (81/19) copolymer that was annealed at 120 C (close to the T c ) for 16 h the T c increased to 128 C but no change of T m was observed. In contrast, the T c increased to 125 C and T m increased to 151 C aer annealing at a higher temperature of 140 C for 16 h. 81 The changes of T c were associated with changes in the Gibbs free energies in the orthorhombic b-phase and hexagonal a-phase during annealing. Annealing of the material below its T c favors the removal of gauche defects in the ferroelectric phase which increases the degree of crystallinity and b-phase concentration. Annealing above T c facilitates the transformation of the paraelectric aphase into the b-phase. The b-phase concentration in the as-cast lm increased from 75% to 93% aer annealing at 130 C for 2 h (Fig. 8). The increase in b-phase concentration led to higher piezoelectric activity and increased the d 33 piezoelectric coefficient from an original value of 13 pC N À1 to 18 pC N À1 aer 4 h of annealing with a three-fold increase in elastic modulus and 10-fold reduction of oxygen permeability. 81 For energy harvesting applications high piezoelectric coefficients, such as d 33 , are benecial, see eqn (2)-(4).
In the case of P(VDF-co-TrFE) copolymers with a VDF content in the range of 65-82 mol%, the T c shows a large thermal hysteresis, it is lower during the cooling process (T Y c ) compared to the heating process (T [ c ). For the P(VDF-co-TrFE) 75/25 mol%, the T Y c ¼ 57-79 C was much lower than that of T [ c ¼ 124 C. 35 Due to the large difference in the free energy between the hexagonal a-phase phase and orthorhombic b-phase phase at T Y c , the phase transition proceeds rapidly even when the lm is cooled slowly. Therefore, TGTG 0 sequences in the hexagonal aphase phase are quenched partly in the orthogonal direction as conformational defects. When annealed above T c , P(VDF-co-TrFE) copolymers undergo a thickening of crystallites, resulting in a strong increase in crystallinity, up to 90% or more for a VDF content in the range 70-80%. 35,93,94,[94][95][96] The T c decreases as the TrFE content increases, and vanishes for P(VDF-TrFE) 50/50, due to cooperative movements in the ferroelectric b-phase near the transition temperature. 87 A maximum b-phase fraction of 66.3% was obtained from the sequential treatment of "annealing and cooling-pressing-electrical poling" while control materials only exhibited a b-phase fraction of 45.3%. 97 For a copolymer crystallising from the melt, the T c was affected by the cooling rate, suggesting that a higher cooling rate leads to VDF-rich crystalline regions due to the TrFE units having difficulty entering into the crystal lattice. In contrast, quenching the material from the melt leads to a broader endotherm, similar to dimethylformamide (DMF) crystallized samples annealed at 120 C. 93 Melt processing, in particular extension ows, also affect the crystallization and chain orientation of polymers. For example, in P(VDF-co-TrFE) copolymers, the extensional ow at a normal stress level of 6.35 Â 10 5 Pa produced an all-trans crystal structure in the 66/34 mol% copolymer. A 7 C higher transition temperature and lower melting temperature was observed in the two-dimensional ow rate range of 0.002-0.03 cm 2 s À1 for the 75/25 mol% copolymer. 98 In both PVDF and P(VDF-co-TrFE) copolymers, the a-phase tends to form when cooling from high temperature and at high cooling rates. The presence of increasing amounts of TrFE increases the formation of b-phase. A lower fabrication temperature favours the formation of polar band g-phases. 99 When an as-cast P(VDF-co-TrFE) (80/20) lm was heated to 200 C for 4 h, then quenched at 100 C, a 43% degree of crystallinity was obtained and the a-phase dominated; when quenched at À20 C, a 56% degree of crystallinity was obtained, and almost 100% b-phase was formed. Therefore, the annealing process affects the local distribution of the TrFE comonomer units in the crystalline lattices, which is reected by the varied T c and crystallinity. It is largely accepted that b-phase melting occurs in the range 165-172 C; a-phase crystals in the range 172-175 C with the g-phase melting between 175 and 180 C. 100 The ferroelectric behaviour of PVDF based materials have been investigated as a function of temperature and poling frequency, as shown in Fig. 9 and 10. 101 For a uniaxially stretched P(VDF-co-TrFE) (50/50) lm, paraelectric behavior was obtained due to the nucleation of electric eld-induced ferroelectric nanodomains inside the paraelectric matrix when it was poled at a high poling frequency (1 kHz) at 100 C and above the T c of 64 C. These ferroelectric nanodomains were highly reversible and could be rapidly depolarized upon removal of the poling eld, as shown in Fig. 10C. At an intermediate poling frequency (10 Hz) at 100 C, an 'antiferroelectric-like' behaviour was observed, which could be attributed to the competition between depolarization and polarization elds upon reversed poling, this corresponds to the behaviour in Fig. 10B. Finally, at a low poling frequency (1 Hz) at 100 C, conventional ferroelectric behaviour with a rectangular hysteresis loop was observed since the small, reversible ferroelectric domains have sufficient time to grow into large irreversible ones. For energy harvesting applications the ability to pole the material and achieve a high remnant polarisation (P r ) which is modulated with stress or temperature is of interest for piezoelectric or pyroelectric harvesting, as in Fig. 10A. For energy storage applications, the antiferroelectric-like ( Fig. 10B) or paraelectric ( Fig. 10C) behaviour is more desirable due to the ability to store and recover energy, as given by the larger areas in the D-E f loops which are indicated by the purple areas in Fig. 10.
A b-phase content of 74% and an overall crystallinity of 42.6% was achieved for melt-quenched stretched PVDF lms. 102 A higher b-phase content of 82% and a d 33 of 21 pC N À1 was obtained by stretching PVDF lms that were solution-cast from N,N-dimethylacetamide (DMAc) solutions. 103 It is reported that an overall crystallinity of 52-60% with a b-phase fraction of 53% was present in PVDF lms prepared by spin coating from an acetone/DMF solution and then stretching uniaxially. 104 The pyroelectricity of a ferroelectric VDF-oligomer [CF 3 (CH 2 CF 2 ) 17 I]evaporated lm was investigated utilizing a low-frequency sinusoidal heat source. 72 The pyroelectric coefficient (p) of the 600 nm-thick VDF oligomer lm was À68 mC m À2 K À1 at 37 C, which is larger than those reported for ferroelectric polymers. The VDF oligomer is therefore a promising material for pyroelectric thin-lm infrared detectors and pyroelectric harvesting.
2.2.4 Nanoconnement. Nanoconnement has recently been used to facilitate the formation of polar b-phase. PVDF nanowires can be predominantly crystallized into the b-phase by connement in a nanoporous structure (e.g. in 200 nm channels), leading to the formation of well aligned polymer chains and crystallites arranged perpendicularly to the channel walls. 105 PVDF nanowires exhibited a higher remnant polarization (P r ¼ 19 mC cm À2 ) than the saturation polarisation (P s ¼ 9 mC cm À2 ), which was attributed to the combination of the polarization of the ferroelectric polymer with the charge derived from the superposition of leakage current to the displacement current. In contrast, thin lms of PVDF without any poling stage did not show any ferroelectric behaviour. P(VDF-co-TrFE) (70/30) nanowires formed by templating exhibited a P r ¼ 7.4 mC cm À2 and P s ¼ 9.6 mC cm À2 , that are similar to as-prepared P(VDF-co-TrFE) thin lms. Maximum d 33 values of À8.2 and À6.5 pm V À1 were obtained for the P(VDF-co-TrFE) and PVDF nanowires. Non-poled P(VDF-co-TrFE) thin lms had a d 33 of À15 pC N À1 , while in comparison, poled lms and bulk materials of both P(VDF-co-TrFE) and PVDF had a d 33 in the range of À20 to À30 pC N À1 (or pm V À1 ).
A signicant change in the crystallisation behaviour of P(VDF-co-TrFE) was observed when conned in a nanoporous template with pores less than 40 nm in diameter, 38 as compared to the bulk material. The copolymer crystallised into an oriented ferroelectric crystal lamellae perpendicular to the pore axis. Both melting and crystallization temperatures decreased with a decrease in pore diameter, and the T c was weakly affected. The presence of ferroelectric phase was preserved down to pore sizes as small as 15 nm. The results imply that nanoconnement enhances the formation and orientation of the ferroelectric b-phase and can enhance ferroelectricity and piezoelectricity in nanoscale P(VDF-co-TrFE) materials.
For a P(VDF-co-TrFE) copolymer with a composition ratio of 72.2/27.8 mol%, 88 the polymer chain length affects the conformations. It was found that a short polymer chain length leads to a higher crystal size and promotes a higher b-phase content; the d 33 piezoelectric coefficient was enhanced by decreasing the molecular weight (M w ) of the copolymer. A maximum d 33 value of À50 pC N À1 was achieved for the composition 72.2/27.8 mol% with a molecular weight of 470 kg mol À1 . Interestingly, the pyroelectric properties were enhanced for the lowest polymer crystalline grain size studied. A pyroelectric coefficient of 37.8 mC m À2 K À1 was obtained with the composition 71/29 mol% with a molecular weight of 505 kg mol À1 . These differences may be related to how the polarisation changes with either stress (thereby inuencing the piezoelectric coefficient) or temperature (thereby inuencing the pyroelectric coefficient).
When a PVDF based material is crystallised and conned in two-dimensional (2D) cylindrical anodic aluminium oxide (AAO), the crystal c-axis becomes perpendicular to the porous cylinder axes, which favours dipole switching. The polarity of the nanopore surface and the surface hydroxyl groups on the template nanopores promote the formation of the polar phase, with a b-phase content of 40.5%. As shown in Fig. 11a, the use of an oxygen plasma treatment increases the number of negatively charged OH À and O 2À groups on the surface of the Al 2 O 3 nanopore template, thereby increasing its hydrophilicity compared to the pristine template; see Fig. 11a and b.
Therefore, the presence of highly polar surface increases the template interaction with the PVDF dipoles, and forces the polymer chains to arrange in a TT conrmation, leading to a polar b-phase fraction of 40.2%. The use of a 3-aminopropyltrimethoxysilane (APMS) treatment increased the hydrophobicity of the nanopore surface, and enhanced the interfacial interactions with the polymer chains, and enhanced the polar phase formation to 71%, as shown in Fig. 11c. 106 However, no polar phases have been obtained from 2D connement down to 35 nm pores aer melt recrystallization.
When recrystallized from the melt in conned 3D polystyrene nanospheres 180 nm in diameter, PVDF exhibited both polar band g-phases, rather than the kinetically favoured aphase, 107 see Fig. 12. These results indicate that PVDF can nucleate homogeneously at a high crystallization rate in the 3D nanoconned space, which does not rely on the polarity of the substrate. The use of 3D nanoconnement is thought to be more effective than 2D nanoconnement in producing polar crystalline phases in PVDF.
By varying the quenching temperature, the crystal structure and properties of PVDF can be tailored for different applications, as shown in Fig. 13. 108 For solution-cast lms, when quenched below 0 C, more b-phase was formed which was aligned to create a self-polarised material with a high d 33 of $49.6 pm V À1 (see Fig. 13c). This is much higher than typical values of À20 to À35 pm V À1 produced by mechanical stretching and poling. These values are comparable to PVDF nano-bres which show a d 33 coefficient of À54 pm V À1 , which originates from the high g-phase content with $75% crystallinity. 109 The high d 33 of the lm was attributed to the high ($100%) b-phase content and high crystallinity of 56%, as shown in Fig. 13a and b. Fast quenching at a low temperature induced a strong thermal eld gradient, which can cause crystals to align along the thermal eld direction. The interaction with polar water can also be a possible reason for the alignment of the b-crystals. 109,110 The self-polarised b-phase and high piezoelectric coefficient of the PVDF lms make them suitable for energy harvesting applications, such a vibration harvesting (Fig. 13d) and examples of devices are described at the end of the review (Section 3). Further details of ferroelectric polymer nanostructures under connement has been described 111 and recently nanostructured arrays of PVDF based polymers have been formed by nanoimprint lithography. 112 2.2.5 Surface charge and nucleation. The nucleation behaviour of a polymer depends on the properties of the nucleating agents, such as surface charge, surface area, lattice matching, concentration and dispersion, as well as the interfacial interactions between the nucleating agents with PVDF chains. 64 Nucleating agents reduce the nucleation energy barrier, increase crystallization kinetics, crystallisation temperature, melting temperature, and the degree of crystallinity. To evaluate the effects of surface charge on the nucleation efficiency, nucleating agents with a positive charge (phosphonium, pyridinium, pyrrolidinium, ammonium, and sulfonium salts), negative charge (sulfate and phosphate salts) and neutral agents (avanthone) have been melt-compounded with PVDF. 64 The addition of 0.5 wt% of nucleating agent altered the crystallization behaviour of PVDF, especially when the melting temperature of the nucleating agent was close to the melting temperature of PVDF. Only the a-phase was formed in the absence of nucleation agents with negative or neutral nucleation agents, while band g-phases were formed in PVDF with positively charged nucleation agents. Fig. 14 shows examples of the chemical structures of positive, negative and neutral nucleating agents.
Nanoparticles can also act as nucleating agent for polymers by increasing the crystallisation kinetics and degree of crystallinity. For PVDF composites lled with Na(M)Y zeolites, the negatively charged zeolite framework induces the formation of the polar g-phase in PVDF when prepared by solution-cast and melt compression. 114 The use of Na(Y) in the composite induced 100% of g-phase in PVDF due to the negatively charged iondipole interactions between the zeolite framework and the PVDF. The exchangeable (M) ion size in the zeolite also affects the PVDF phase structure. The presence of small sized lithium (Li) ions increased the crystallisation of polar phases, as compared to Na(Y), while the presence of potassium (K) and cesium (Cs) has the opposite effect. This behaviour depends on the interaction competition between the alkali ion-zeolite structure and alkali ion-molecular dipoles. Using larger sized ions and stronger interactions with zeolite structures reduced the interactions with polar polymer chains, leading to a lower fraction of polar phase. The relative permittivity ranged from 47 to 3 for PVDF/Na(Li)Y and PVDF/Na(Cs)Y, respectively, while the electrical conductivity of PVDF/Na(Li)Y was three orders of magnitude higher than that of PVDF/Na(Cs)Y. An increased relative permittivity can oen be related to increased conductivity in the material.
With the addition of graphene oxide (GO) nanoplatelets to PVDF, 115 only the g-phase was induced to form in PVDF when crystallized from the solution, and only a-phase forms from melt crystallization. When GO was combined with a positively charged surfactant (cetyltrimethylammonium bromide, CTAB), more g-phase crystals were formed in PVDF during isothermal melt crystallization at 160 C for 80 min. It is suggested that there are two distinct stages during the melt crystallization of PVDF/GO composites in the presence of CTAB, i.e., a simultaneous growth of gand a-phases, which is followed by an ato gphase transition. The T m was increased by 10 C for the g-phase PVDF/GO composites. The addition of CTAB was thought to strengthen the ion-dipole interactions between the GO and PVDF, as compared to weak p-dipole interaction between GO and PVDF.
Similarly, the effects of the surface charge on the crystal structure of PVDF were studied using nanoparticles of CoFe 2 O 4 whose surface was treated with three types of surfactants, i.e., anionic (SDS), nonanionic (Triton X-100), and cationic (CTAB). 116 A higher fraction of polar b-phase was obtained when CoFe 2 O 4 nanoparticles with a negative electrostatic charge were added. The b-phase content reached 30% and 90% for CoFe 2 O 4 / PVDF and CoFe 2 O 4 -SDS/PVDF, respectively, with a resultant d 33 of 23 and 33 pC N À1 respectively. This behavior was attributed to the interaction between the negatively charged magnetic CoFe 2 O 4 particles and the positive polymer CH 2 groups.
The ion-dipole interactions are described as (i) negatively charged surfaces with the CH 2 dipoles in PVDF, or (ii) the positively charged surfaces with the CF 2 dipoles in PVDF. By comparing the effects of positively-and negatively-charged organic nucleation agents, 113 as in Fig. 15, the positively charged nucleation agents interact more strongly with the partially negative CF 2 dipoles of PVDF, resulting in a lower nucleating energy barrier and increased crystallization kinetics and crystallization temperature for PVDF; this induces the formation of polar (b, g) phase formation. The addition of positively charged organic molecules in solid PVDF induced a 100% of g-phase formation, while the b-phase PVDF becomes dominant when in the molten state. Almost 100% of polar phases were formed by the addition of 3 wt% of positively charged small molecules (1butyl-3-methylimidazolium hexauorophosphate). The band g-phases can give rise to ferro-, pyro-, and piezoelectricity for harvesting, and the g-phase can lead to increased transparency 117 and exibility 113 of PVDF.
In addition to tailoring the processing conditions and heattreatment conditions, recent approaches to tailoring the piezoand pyro-electric properties are to use polymer blending and form nanocomposites, which will be now discussed. 121,122 For example, the miscibility of PVDF with amorphous PMMA is driven by hydrogen bonding between the carbonyl group of PMMA and CH 2 of PVDF and dipole-dipole interactions between CH 2 of PMMA and CF 2 of PVDF. 119 The blending of amorphous PMMA with PVDF decreased the crystallinity and crystal size of PVDF which were mostly in the polar b-phase when the PMMA content was above 25 wt%. 118 With the assistance of rigid PMMA segments, the b-phase was easier to pole and was reversible with electric eld changes. A higher PMMA content ($40 wt%) and quenching process favoured the ato bphase transition. Therefore, PVDF/PMMA blends with a high bphase content exhibited a high relative permittivity, and a low dielectric and energy loss due to the presence of PMMA. 118 Semicrystalline PBS is miscible with PVDF, and can promote the formation of b-phase in PVDF, depending on the PBS content and quenching temperature. 120 The b-phase fraction increased with increasing PBS content, up to 50 wt%, while the addition of greater amounts of PBS can restrict the nucleation and growth of the b-phase, thus resulting in more a-phase. Lowering the quenching temperature benets the formation of b-phase in PVDF.
Semicrystalline PA11 has comparable coercive elds and remnant polarization with PVDF, but a low piezoelectric response (<4 pC N À1 , see Table 1) at room temperature because of its higher glass transition temperature (ca. 50 C) compared to PVDF ($À30 C). 123,124 When blending ferroelectric PA11 and PVDF, the glass transition temperature and melting point of PA11 decreases with increasing PVDF concentration, due to the dipolar intermolecular interactions between the polar amide groups (-NH-CO-) in PA11 and the polar -CF 2 groups in PVDF. 121,125 It has also been found that the hydrogen-bonded structure of PA11 becomes more disordered as the PVDF concentration is increased. The PVDF developed a larger proportion of polar band g-phase in blends with a high PA11 concentration compared to pure PVDF under similar melt quench conditions. 121 During the uniaxial drawing process, the phase transformation of PVDF from the nonpolar a-phase to the polar b-phase is more complete, with more ordered b-crystals than in pure PVDF. These structural changes and the dipolar interactions between PA11 and PVDF lead to a 30% increase in piezoelectric properties and improved high-temperature stability of the blends at temperatures up to 160 C. 121 These properties enables this new polymeric blend material to be used in electroactive applications, such as sensing and harvesting in more hostile environments.
The formation of unique orientation textures due to conned crystal growth of PVDF in PVDF/PA6 blends of drawn lms have been reported. 122 Uniaxial-drawn PVDF/PA6 blends were heat-treated at 180 C for 3 minutes to melt the PVDF component, followed by non-isothermal crystallization of PVDF at a cooling rate of 0.5 C min À1 . For PVDF/PA6 as a 50/50 blend, stretched domains of PVDF with a diameter of 0.2-0.5 mm were dispersed in a PA6 phase. Spatial connement of the crystal growth resulted in the alignment of the crystal b-axis along the long axis of the domains, since the PVDF crystallized as thin cylindrical domains. The orientation behaviour is different from the oriented crystallization of PVDF/PA11 125 in which trans-crystallization from the interface causes an a-axis orientation in the drawing direction. It is thought that the domain size inuenced the mechanism of oriented crystallization.
A recent study showed that a P(VDF-co-HFP)/poly(ether imide) blend with a poly(ether imide) content larger than 80% created more free volume in the polymer matrix, thereby providing more space for dipole orientation polarization and providing an enhancement of the relative permittivity of the polymer blends. 126 2.2.7 Formation of nanocomposites. Polymer-based nanocomposites have attracted interest for high energy density capacitors with high energy density (U e ), 19 since these heterogeneous materials combine the high relative permittivity and dielectric properties of nanollers, such as ferroelectrics, with the high breakdown strength, low dielectric loss, and lightweight nature of polymeric matrices.
As discussed, PVDF and its copolymers contain mainly of aphase ($40%) 127 or a trace amount of b-phase when cooled from the melt; Fig. 4. A signicant body of research has demonstrated that nanoparticles with a negatively charged surface can interact with the positively charged -CH 2 groups, and induce the formation of the polar b-phase, with fractions up to 100%, 64 140 ), and conducting polymers. The effect of nanoparticle additions on the structure and piezoelectric properties of PVDF polymers generally depend on their nucleation efficiency, supercooling effect and interfacial interactions. The nucleation effect of the nanoparticle additions strongly depends on the particle size, 129,135 shape, 141 surface chemistry, 135 concentration, 130 dispersion, interfacial interaction and processing conditions.
2.2.7.1 Ceramic particles in nanocomposites. As described in Section 1, the generation of a piezoelectric potential under strain is due to the displacement of the positive and negative charges in the b-phase of PVDF. With the incorporation of ferroelectric inorganic particles, such as BaTiO 3 , the piezoelectric potential can be further enhanced. 129 BaTiO 3 hollow nanospheres 141 with particle sizes of z20 nm and surface area of 297 m 2 g À1 were surface-treated to enhance their compatibility with PVDF. Both the large specic surface area and increased surface functionality of the hollow spheres increased the b-phase content and degree of crystallinity at 16 wt% of ller. The changes in crystallinity led to a high relative permittivity of 109 and high energy density (U e z 21.7 J cm À3 ) with a high retained breakdown strength (E b ¼ 3.81 Â 10 3 kV cm À1 ) compared to pristine PVDF (3 0 z 11.6 and U e z 2.16 J cm À3 at 3.98 Â 10 3 kV cm À1 ). Surface functionalised Ba 0.6 Sr 0.4 TiO 3 nanobers 142 showed good dispersion and strong interfacial adhesion with PVDF matrix, which resulted in high relative permittivity (3 0 $ 12) and high breakdown strength (3900 kV cm À1 ) for the composite containing 2.5 vol% of nanobers. The maximum energy storage density reached 7.5 J cm À3 for a PVDF nanocomposite under the electric eld of 3900 kV cm À1 , which is three times that of pure PVDF (2.8 J cm À3 at 4000 kV cm À1 ). A recent study on BaTi (1Àx) Zr x O 3 (BTZO) nanocube lled PVDF composites for harvesting reported that 143 the exible PVDF/ BTZO composite lms exhibit a high electrical output up to $11.9 V and $1.35 mA compared to PVDF/BaTiO 3 lms which had an output of 7.99 V and 1.01 mA when subjected to a cyclic stress at 21 Hz with a constant load (11 N). The doping of various amount of Zr 4+ (x ¼ 0, 0.05, 0.1, 0.15, and 0.2) into the Ti 4+ site of BaTiO 3 can further enhance the piezoelectric performance of the particles. The piezoelectric potential of the PVDF/BTZO composites was adjusted by varying the composition ratio of BTZO and PVDF, electrical poling and mechanical loading. The composite generators were used to measure different water velocities at an outlet pipe and the peak power of the piezoelectric nanogenerator varied from 0.2 to 15.8 nW for water velocities ranging from 31.43 to 125.7 m s À1 .
2.2.7.2 Metal and metal salts in nanocomposites. For metal and metal salt lled PVDF composites, it was shown that 5 wt% of copper induced the formation of up to 90% of b-phase due to the high interfacial area between the well-dispersed nanoparticle surface and the polymer. 135 Superior ferro-and piezoelectret properties in a self-poled, porous hybrid ferroelectretic polymer nanocomposite lm was achieved by in situ generation of platinum nanoparticles embedded in a P(VDF-co-HFP) matrix. 134 The cooperative functionality between the self-polarized b-phase and the micropores as charge trapping sites was achieved by using a simple solvent evaporation method. The resulting porous hybrid lm exhibited a square-shaped hysteresis loop with large remnant polarization (P r z 61.7 mC cm À2 ), high piezoelectric charge coefficient (d 33 $ À686 pC N À1 ), and high dielectric properties (relative permittivity 3 0 ¼ 2678 and tan d ¼ 0.79 at 1 kHz); this high permittivity may be associated with the high loss (tan d). An open-circuit output voltage of 18 V with a 17.7 mA short-circuit current were generated under a 4 MPa stress. The high piezoelectric energy conversion efficiency (h piezo z 0.2%) of the nanogenerator demonstrated its potential for piezoelectric-based energy harvesters.
The addition of metal salts, such as cerium(III)/yttrium(III) nitrate hexahydrate (1-30 wt%), 144 or Fe 2 O 3 -Co 3 O 4 nanoparticles 138 to PVDF induced the formation of b-phase due to a strong ion-dipole interaction through the formation of hydrogen bonds between the water molecules of the salts and the -CF 2 dipoles of the polymer chains; this led to a large permittivity. A 15 wt% SiO 2 -loaded PVDF lm exhibited a high permittivity, due to the homogeneous dispersion and interfacial interaction of SiO 2 nanoparticles in the PVDF matrix. 131 For a P(VDF-co-HFP) matrix lled with mesoporous SiO 2 nanorods, the anisotropic shape of the SiO 2 nanorods and ordered mesopores doubled the amount of the -OH groups present. This increased the intermolecular interactions and enhanced the bphase content and ferroelectric properties of P(VDF-co-HFP). 145 Using this approach, the nucleation of the ferroelectric b-phase in PVDF can be inuenced by the geometry of the llers through the interface interactions between the local electric eld of the ller and PVDF dipoles, such as ion-dipole and dipole-dipole interactions.
Ferroelectric P(VDF-co-CTFE) terminated with phosphonic acid groups were synthesised and subsequently coupled with ZrO 2 llers. 136 The functional chain end-groups can form covalent coupling with the ZrO 2 surface, thereby providing the nanocomposites with stability and a uniform ller dispersion. The presence of 9.1 wt% ZrO 2 increased the crystallization temperature of the copolymer from 82 to 91 C, and increased the crystallinity from 18.3 to 22.8%. As a result of the intimate coupling between the ller and matrix, the interfacial interaction regions between the polymer and ZrO 2 increased the energy density at high electric elds. The energy density reached a maximum of 11.2 J cm À3 at 270 MV m À1 with 9.1 wt% of ZrO 2 , this was a 60% increase in comparison to the copolymer matrix. The improvement in the energy storage capability of the nanocomposite was ascribed to changes in polymer microstructure and an increase in crystallinity, the interfacial region and the rise of the electric displacement induced by the incorporation of the nanollers.
The incorporation of a cerium(III)-N,N-dimethylformamidebisulfate [Ce(DMF)(HSO 4 ) 3 ] complex 140 into PVDF provided a higher yield (99%) of the polar band g-phases. An enhancement of the output voltage ($32 V) of a nanogenerator based on a non-electrically poled cerium(III) complex containing PVDF composite lm was achieved by repeated human mechanical loading, whereas pure PVDF did not show any response. This high electrical output was due to the electrostatic interactions between the uoride of PVDF and the positive charge cloud of the cerium complex via H-bonding and/or a bipolar interaction between the opposite poles of the cerium complex and PVDF, as shown in Fig. 16a-c. The capability of the composite lm to charge a capacitor shows its capability as a piezoelectric-based energy harvester. The cerium(III) complex doped PVDF composite lm exhibited an intense photoluminescence in the UV region, which may be due to participation of an electron cloud from the negative pole of the bipolarized PVDF. This fact may also be of interest for the development of exible solid-state UV light emitters, as shown in Fig. 16d. 2.2.7.3 Nanoclay additions. Nanoclay, in particular, layered montmorillonite has been shown to provide excellent reinforcement and barrier functions to polymers. A variety of organoclays modied with cationic surfactants have been investigated for modifying PVDF-based polymers. Commercial organoclays, such as Cloisite®Na, Cloisite®6A, Cloisite®15A, Cloisite®20A, Cloisite®25A, Cloisite®30B, Lucentite STN, Nanocor Inc I.34TCN have been studied. The main difference between the organoclays above are the different surface treatment and polarity. It was found that the organoclay promotes the formation of b-phase of PVDF, while unmodied clay shows little or no inuence. Solution-casting 146 has been shown to facilitate dispersion and strengthening of the interaction between the organoclay and PVDF, as compared to meltprocessing.
The effects of nanoclay inclusions on the crystallisation behaviour of PVDF depend on the dispersion, exfoliation and interfacial interactions of nanoclay in the PVDF matrix. Three types of organoclay modied by different surfactants were studied: ammonium (C18), pyridinium, and phosphonium. It was found that the three types of organoclay could be readily dispersed in PVDF melts. The ammonium-clay showed the best dispersion and phosphonium clay was more efficient in forming the polar b-phase. 127 High b-phase fractions of $99% were obtained at 5 wt% of octadecyltriphenylphosphonium bromide modied clay with a 28.2 wt% organic content. In comparison, the addition of 5 wt% of unmodied clay leads to only 23% of bphase in the composites. The presence of nanoclay also acted as a nucleating agent and enhanced the melting and crystallization temperature of PVDF by 10 and 13 C, respectively. For the composite containing 3 wt% of nanoclay, the relative permittivity at 1 kHz was approximately 9.6 with a dielectric loss less than 0.05, the P r was 6.3 mC cm À2 , as compared to 5.0 mC cm À2 for pristine PVDF. A higher loading of nanoclay, over 5 wt%, led to a higher dielectric loss, increased leakage current and early breakdown due to the aggregation of the clay in the PVDF matrix. By tuning the surface charge and dispersion of the nanoclay in PVDF, the energy density of the PVDF/nanoclay composites was increased from 5.34 to 5.91 J cm À3 at 1 wt% clay content and the highest energy density could reach 10.2 J cm À3 . 147 In addition to promoting b-phase formation, organoclay platelets can also stabilize the b-phase from depolarisation. [148][149][150] The organoclay may play three roles in inuencing the crystal structure and properties of PVDF: 68,149 (i) a supercooling effect, (ii) a heterogeneous nucleating effect and (iii) intermolecular interactions between the polymer chains and silicate sheets. To investigate the role of organoclay, solutioncast PVDF/organoclay (Lucentite STN) composite lms with different thermal treatments were studied. This included annealing at 160 C/24 h, melt-quenching followed by annealing treatment 160 C/5 h and melt-slow cooling (10 C min À1 ). The as-cast samples exhibited nearly pure g-phase irrespective of the organoclay content, the melt-slow cooled samples showed a-phase at lower clay concentration, and a mixture of aand gphases at a higher clay content. The samples prepared by meltquenching followed by annealing had a mixture of a-, band gphases across a range of clay contents. Upon heating, the bphase initially transformed to the thermodynamically stable gphase before melting. The increased melt-crystallization temperature of PVDF with the addition of organoclay indicated that the clay did not show any super-cooling effect. Ultra-thin lms of PVDF/organoclay (Lucentite STN) nanocomposites have been prepared by heat-controlled spin coating. 71 The Lucentite STN favours the formation of b-phase and orientation in nanoscale thin lms, irrespective of preparation temperature, which leads to a high remanent polarization. In comparison, thick PVDF/organoclay nanocomposites lms only contained aphase aer quenching and slow-cooling from the melt, while the nanoscale thin nanocomposite lms showed a mixture of band g-crystalline phases without any a-crystalline phase. Therefore, the sample thickness also affects the crystal structures of the PVDF composites.
Organoclay platelets can retard the relaxation of the PVDF chains and stabilize conformation due to the presence of iondipole interaction between the exfoliated nanoclay layers and the PVDF. An increase of organoclay concentration should therefore increase the level of interaction. For organically modied montmorillonite (OMMT) modied PVDF, the a-phase dominates when the OMMT concentration is below 0.025 wt%, while the b-phase fraction exceeds the a-phase fraction when the OMMT concentration is between 0.025 and 0.5 wt%. 151 It is found that an increase of organoclay above 0.5 wt% introduces gauche defects in the all-trans conformation and results in a mixture of band g-crystals in the polymer. 151 In this case, the interactions between the clay and PVDF is responsible for crystal modication rather than the heterogeneous nucleating actions and super-cooling effect. 63,148,152 The presence of a second polymer which is compatible with PVDF can also benet the formation of the polar ferroelectric phase. 153 In addition to the well-studied PVDF/PMMA blends, as described in Section 2.2.6, the addition of a polymeric compatibilizer such as acrylic rubber (ACM) can facilitate the intercalation and dispersion of nanoclay in the PVDF matrix, and promote polar band g-phase formation. 153 A relative permittivity of 16 (at 10 Hz) was obtained for PVDF/ACM/clay (90/10/5 wt%), which was 40% higher than that of a PVDF/clay (100/5 wt%) nanocomposite without ACM.
Therefore, organically modied clays generally have good dispersion and strong interfacial interaction with PVDF. Exfoliated organoclay nanosheets promote and stabilise the epitaxial growth of the b-phase PVDF 149,154 mainly by the stronger interfacial interactions rather than heterogeneous nucleating effects or super-cooling effect. 149 The PVDF/clay nanocomposites have mainly b-phase, and also have g-phases coexisting at low crystalline temperature (T c < 155 C), the coexistence of gand b-phases was found at a high crystalline temperature range (T c > 155 C). The b-PVDF nucleation is inuenced by the geometrical factors, interactions at the interface between the nanoparticles and the PVDF dipoles and particle concentration. 31,32, 34 2.2.7.4 Carbon nanotubes additions. Single-walled (SWCNT) and multiwalled carbon nanotubes (MWCNT) have been used to modify PVDF and its copolymers. Utilizing a solution-mixing method using dimethylacetamide (DMAc) as the solvent, 155 pristine MWCNT (diameter 10-50 nm, length 4-10 mm) induced the formation of both aand b-phases in PVDF under sonication, while no b-phase was formed in mechanically mixed mixtures. According to density functional theory, more energy is required to form the TT b-phase, and more TGTG 0 a-phase can transform to the TT conformation under sonication, and the TT molecular chains can bind the CNT surface more tightly than the TGTG 0 polymer chains.
For SWCNT lled P(VDF-co-TrFE) composites prepared by solution-casting followed by annealing at 70 C for 38 h, 156 the pyroelectric coefficients of the composites were higher than that of pure P(VDF-co-TrFE) copolymer. The increase in the pyroelectric coefficients of the composites as a function of temperature is more gradual compared the copolymer, as shown in Fig. 17a. This may be due to the interfacial interactions between the SWCNTs and the polymer chains which hinder the mobility of the dipoles at higher temperatures, thereby limiting the overall contribution to pyroelectricity. The measured d 31 coef-cient was 25 pC N À1 , which is higher than the 20 pC N À1 for the pure PVDF-TrFE lm with no additions, see Fig. 17b.
By directly melt-compounding PVDF with pristine MWCNTs of diameter of 20-40 nm and length 5-15 mm through a twinscrew extruder, Yuan et al. 157 reported the relative permittivity of PVDF/MWCNTs composites to be as high as 3800, which is three orders of magnitude higher than pristine PVDF, while maintaining a low conductivity level at 10 À5 S m À1 . The enhancement was explained by a reinforced Maxwell-Wagner-Sillars effect due to the wrapping of PVDF chains on the MWCNTs surface and increased interfacial interactions, but the effect of the pristine MWCNTs on the piezoelectric active phase of PVDF was not reported and is of interest for further study.
Without additional post-treatment, only a trace amount of bphase PVDF could be formed in PVDF/CNT nanocomposites when processed by melt-cooling 158,159 and melt-spinning. 160 One of the benets of the inclusion of nanoparticles in PVDF is that the crystalline structures of PVDF can be tuned using surface modied particles, without additional annealing or electrical poling treatment. The nucleating efficiency of three types of surface functionalised MWCNTs in b-phase PVDF were compared. 159 The contents of the functional groups on the MWCNTs surface were carboxyl (-COOH $ 3 at%), amino (-NH 2 $ 0.5 at%) and hydroxyl (-OH $ 1.2 at%), respectively. It was demonstrated that the NH 2 -MWCNTs induced the highest percentage of b-phase (17.4%) in PVDF, 159 followed by OH-MWCNT (11.6%) and unmodied MWCNTs (9.4%). The nanocomposites containing COOH-MWCNTs had the lowest amount of b-phase (4.7%). It is believed that the combined effects of the dispersion of MWCNTs and the interfacial interactions account for the formation of b-phase in PVDF, as shown in Fig. 18. The NH 2 -MWCNTs and COOH-MWCNTs were meltblended with PVDF/PMMA blends, 161 and only the NH 2 -MWCNTs favoured the formation of b-phase, where both the pure blends and COOH-MWCNTs lled composites exhibited only b-phase aer melt-mixing. This was ascribed to the Ionic liquids are a good dispersant for CNTs. It was found that the addition of 2 wt% of pristine MWCNTs only induced 6.1% b-phase in PVDF 162 aer cooling from the melt. However, when the MWCNTs were modied with an ionic liquid (1-butyl-3-methylimidazolium hexauorophosphate [BMIM] + [PF6] À ), almost 100% b-phase was achieved in PVDF. This was thought to be due to the interactions between the planar imidazolium cations of the ionic liquid that were wrapped on the MWCNTs surface with the -CF 2 bonds of the PVDF, thereby improving the dispersion of MWCNTs in the PVDF melt. In comparison, the incorporation of only a ionic liquid depressed the PVDF melt crystallization due to the miscibility of ionic liquid with PVDF and led to a higher content of polar band g-phase compared to using pristine MWCNTs. This indicates the synergetic effects of MWCNTs and ionic liquids on the b-phase formation in PVDF during melt cooling. MWCNT covalently modied with 3-aminoethyl imidazolium bromide 162 led to the formation of 100% bphase in PVDF at only 1 wt% concentration by both solventcasting and melt-blending processes. 163 It was concluded that covalently linked ionic liquids with MWNTs is an efficient method to induce polar phase formation in PVDF compared to physically modied ionic liquids and MWNTs.
In addition to ionic liquids, MWCNTs modied by ester groups (e.g. -COOC 2 H 5 ) 164 and PMMA 165 have been used to fabricate PVDF/CNT nanocomposites through solution-mixing, in which the fraction of b-phase was highly dependent on the modication of MWCNTs and their dispersion in the matrix. The ester (-COOC 2 H 5 )-functionalized MWCNTs (FMWCNTs) 164 were well-dispersed in the PVDF matrix and the FMWCNTs facilitated the transformation of ato the b-phase by the interaction of the pC]O group in the FMWCNTs and the pCF 2 group of PVDF. However, only a maximum of 50% b-polymorph PVDF was achieved, even at a high loading of the FMWCNT in the melt-cooled samples. PMMA-graed MWNTs via nitrene chemistry could induce the formation of an almost fully b-phase PVDF at a 5 wt% loading 165 from solution-cast PVDF nanocomposites.
For a PVDF/PMMA (50/50) blend, the introduction of a small amount of acidized CNTs (0.2 and 0.5 wt%) accelerated PVDF crystallisation, and two types of spherulites were observed in the PVDF/PMMA blends, namely ring-banded spherulites and compact spherulites. The acceleration of the crystallization of the PVDF phase was attributed to the joint effects of CNT nucleation and inducing phase uctuation between PVDF and PMMA during the crystallization process. 166 As mentioned in Section 2.2.6, the fraction of PMMA in the blends governs the origin of polymorphism in the PVDF, 118 while the effect of steady shear was found more pronounced in the blends rich in a-phase crystals. 167 Composites based on PVDF lled with NH 2 -MWNTs showed two distinct structural relaxations in dielectric loss owing to mobility connement of PVDF chains and smaller cooperative lengths. The aspect ratio of CNTs also affect the nature of the dispersion, nucleation and reinforcement of polymers. Two MWNTs with a similar diameter of 10-20 nm, and different lengths 5-15 mm (L-MWNT) and 1-2 mm (S-MWNT) were mixed with PVDF in DMF solutions. It was found that b-phase structures were formed for L-MWNT at 2 wt%, and a mixture of aand b-phases were detected at below 2 wt% of L-MWNT. For the short S-WMNT, a mixture of aand b-phases coexisted at a concentration at or below 2 wt%, which is believed to be due to the high-aspect-ratio CNT surface having more zigzag carbon atoms, which match with the all-trans conformation of the bphase. 168 Post-treatment can also induce a greater ato b-phase transition and dipole orientation in PVDF nanocomposites. By mechanical stretching, 169 a high phase transformation from ato b-phase ($96%) was achieved for nanocomposites with greater than 1.0 wt% carbon nanobers (CNF). The AC conductivity of CNF/PVDF composites decreased signicantly when the percolation threshold was raised from 1.0 to 4.2 wt% CNFs aer stretching. This was attributed to the reduced crystallinity induced by the phase transformation from ato b-phase as well as the CNF re-orientation.
The thermal conductivity of PVDF is approximately 0.22 W mK À1 , and was increased to 0.47 W mK À1 aer mixing the polymer with 10 wt% of CNT. With the addition of only 1 wt% GO, the thermal conductivity of PVDF/CNT/GO nanocomposites reached 0.95 W mK À1 . In comparison, the thermal conductivity of PVDF/GO with 1 wt% of GO was only $0.27 W mK À1 . Therefore, the presence of low intrinsic thermal conductive GO (0.14-2.87 W mK À1 (ref. 170)) may be of benet for high heat transfer rates in pyroelectric applications; for example eqn (6) indicates a high rate of temperature change are needed to produce high pyroelectric currents. Although the crystallinity of the matrix in the PVDF/CNT/GO composites is decreased in comparison with PVDF/CNT composites, a large number of polar g-phase crystallites were induced. The presence of GO facilitated the dispersion of CNTs and the formation of a denser CNT/GO network structure in the PVDF matrix is thought to be the reason for the enhanced thermal conductivity. 171 PVDF composites containing TiO 2 coated MWCNTs nanoparticles have been prepared through a solution-cast method, 172 followed with mechanical rolling. A highly oriented structure with both PVDF lamella and TiO 2 -MWCNT core-shell structures (TiO 2 @MWCNTs) were formed, and such an aligned structure led to enhanced breakdown strength and a piezoelectric coefficient d 33 of $41 pC N À1 when the particle loading was at 0.3 wt%; this is almost double that of the pure PVDF (see Table 1). MWCNTs were coated with a continuous layer of TiO 2 nanoparticles (TiO 2 @MWCNTs) by a simple hydrothermal process and TiO 2 @MWCNTs/PVDF composites were prepared by solution casting. Compared to the pristine MWCNTs/PVDF composites, the TiO 2 @MWCNTs/PVDF composites had enhanced permittivity and lower dielectric loss. In addition, the breakdown strength of the TiO 2 @MWCNTs/PVDF composites was also improved, which is favourable for enhanced ferroelectric properties and storage. 173 Therefore, the effects of CNT on the nucleation of b-phase of PVDF is mainly dependent on the surface charge-dipole interactions (surface chemistry), the particle size and concentration, as well as processing conditions (solvent, temperature, annealing, shearing, post-treatment).
2.2.7.5 Graphene additions. Graphene and its derivatives are effective nanoparticles for the modication of PVDF and its copolymers. The oxygen functional groups on the surface of GO or reduced graphene oxide (RGO) can interact with the -CF 2 in PVDF via electrostatic interaction and/or hydrogen bonding, thereby beneting the nucleation of ferroelectric gor bphase. 174,175 With a solvent casting route, approximately 100% of the polar ferroelectric b-phase was formed in PVDF/GO lms with a 0.1 wt% GO content, 175 and 80% of b-phase formed in PVDF/ RGO lms, 176 which led to enhanced mechanical properties, relative permittivity and electric polarization. However, PVDF/ graphene nanocomposites oen suffer from higher dielectric loss and lower breakdown strength compared to composites employing insulating piezoelectric ceramics, this is due to the presence of high electric elds in the composite due the presence of electrically conductive ller. 177 The volume fraction of the llers should therefore be as low as possible in order to maintain high breakdown strength and energy density.
Surface modication of graphene or GO provides a route for enhancing the dispersion and interfacial interactions with polymers, thereby leading to higher composite performance at lower ller loadings. To evaluate the effects of surface functionalisation of graphene on the formation of b-phase in PVDF, 178 a thermally-reduced exfoliated graphene (EG) with various oxygen-containing functional groups including uorination-EG, ozone-EG, and PMMA-g-EG were added to PVDF through a solution-mixing method with a 0.5 wt% ller content. The b-phase fraction of PVDF for the variety of systems was PMMA-g-EG > ozone-EG > uorination-EG > EG and was related to its specic interaction between the C]O group of PMMA and the CF 2 group of PVDF. In the frequency range of 10 2 to 10 7 Hz, the relative permittivity of the composites showed a linear relationship with the content of the carbonyl groups of the llers, in the order of PMMA-g-EG > ozone-EG > uorination-EG > EG. Among all the composites studied, the PVDF/PMMA-g-EG composite had the highest relative permittivity (3 0 ¼ 18) at 100 Hz.
When using an ionic liquid (IL) to modify graphene, for example, 1-hexadecyl-3-methylimidazolium bromide, 179 both graphene and the IL play a positive role in the crystallisation of the b-phase in PVDF. This was ascribed to the existence of graphene-cation interaction between the imidazolium cation and the aromatic carbon ring structure, and the electrostatic interaction between the -CF 2 group of the polymer backbone and imidazolium cation. The addition of 3 wt% of IL functionalized GO to PVDF promoted the formation of b-phase, and increased the melting temperature and glass transition temperature. 180 The maximum increase in the storage modulus (73%), Young's modulus (333%) and tensile strength (628%) was at 3% ller concentration. A sharp increase of DCconductivity of the composite to $10 À2 S cm À1 occurred at the percolation threshold of 0.1 wt%. The relative permittivity increased from 3 0 $ 7.5 for pure PVDF to 3 0 $ 13.5 for 3% ller with a percolation threshold at 0.1 wt%. PVDF/NH 2 -treated graphene nanodots (GNDs)/RGO nanocomposites were developed to utilise the synergetic effects of the RGO sheets and NH 2 -treated GNDs. 181 The RGO sheets promoted the formation of b-phase by disrupting the nucleation of a-phase in the PVDF. The -NH 2 groups attached on the surfaces of GNDs to effectively interact with the PVDF, thereby stabilising the polar bor g-phases. The resulting PVDF/NH 2 -treated GND/RGO nanocomposites exhibited a higher relative permittivity (3 0 z 61) and larger energy density (U e z 14.1 J cm À3 ) compared to pristine PVDF (3 0 z 11.6 and U e z 1.8 J cm À3 ).
The addition of GO, magnetic iron oxide (Fe 3 O 4 ) nanoparticles of size 10-12 nm, or the combination of both GO and Fe 3 O 4 led to the transformation of PVDF from the a-phase to the b-phase, which may be due to the heterogeneous nucleating effects of the nanoparticles and interfacial interactions. 182 With the addition of 5 wt% GO and 5 wt% Fe 3 O 4 , the maximum saturation polarisation was 0.065 mC cm À2 , this is higher than PVDF (mainly a-phase, 0.038 mC cm À2 ) and PVDF/Fe 3 O 4 (0.058 mC cm À2 ), but lower than that of PVDF/GO (0.1 mC cm À2 ), which may be due to the conducting nature of Fe 3 O 4 . A higher permittivity was observed for the composite lms (3 0 ¼ 12-19) than the pure PVDF lm (3 0 $ 6), with a dielectric loss of approximately 0.6. The enhanced magnetic, ferroelectric, dielectric, magneto-dielectric coupling and structural properties of the PVDF/Fe 3 O 4 -GO nanocomposite lms were attributed to the homogeneous dispersion and good alignment of Fe 3 O 4 nanoparticles and GO in the PVDF matrix, along with a conversion of nonpolar a-phase PVDF to the polar b-phase.
For RGO-ZnO lled PVDF lms, 183 a ato b-phase transformation was observed and a maximum content of 83% of bphase was determined by FTIR. A decrease in the size of the spherulitic crystal structure of PVDF/RGO-ZnO nanocomposites was observed and a combination of RGO, ZnO and Fe 3 O 4 led to approximately a 50% decrease of b-phase content in the PVDF lms. 176 However, with a Fe-doped RGO, a 99% of polar g-phase was formed in PVDF when the Fe-RGO content was 2 wt%, 184 which was thought to be due to electrostatic interactions among the -CH 2 and -CF 2 dipoles of PVDF and the delocalized p-electrons and oxygen functionalities of Fe-RGO via ion-dipole and/or hydrogen bonding interactions, as shown in Fig. 19a and b. The nanocomposite lms generated an open circuit output voltage and short circuit current up to 5.1 V and 0.254 mA respectively on loading with a human nger as a piezoelectric energy harvester. In addition, the nanocomposite showed a higher electrical energy density of z0.85 J cm À3 at an electric eld of 537 kV cm À1 , higher than that of pristine PVDF of 0.27 J cm À3 , as shown in Fig. 19c and d. This is of interest for combined harvesting and storage systems.
2.2.8 Electrospinning and nanocomposites. Electrospinning has become a promising technique for producing exible piezoelectric polymers as it can induce the formation of more polar crystal phases and align the molecular dipoles with a single processing stage, which is ascribed to the simultaneous mechanical stretching and electrical poling. 185 In addition, other characteristics such as ease of setup, scalability, generation of continuous nanobers and membranes with high specic surface area and high porosity are also benecial for producing energy harvesting devices. A variety of devices, such as high frequency transducers, implanted biosensors, vibration absorbers and composite pressure sensors have been reported. 186 A typical electrospinning apparatus consists of a syringe with a metal needle connected to a high voltage dc power supply. When the polymer solution is ejected through the metal tip at high voltage (7-30 kV), the pendant droplet at the syringe tip becomes electried, and is distorted into a conical shape, known as the 'Taylor cone'. This distortion is caused by the electrostatic repulsion between the surface charges and the coulombic force exerted by the external electric eld. 187 When the electrostatic repulsion between surface charges overcomes the surface tension of the solution, an electried jet of polymer solution is ejected from the syringe tip and deposits onto a grounded collector, while mechanical stretching and electrical poling occur simultaneously during the bre solidication process. Therefore, electrospinning can be a single process to induce preferential orientation and formation of ferroelectric dipoles in PVDF and its copolymers. 188,189 The electrospinning parameters, such as applied voltage and electrode-to-collector distance (from less than 1 cm, up to 20 cm), solvent polarity, polymer molecular weight and concentration, and additives (BaTiO 3 ,190 CNTs,191,192 Ag-CNT, 188 clay, 193 graphene, SiO 2 , and ionic liquid 194 ) have considerable effects on the bre morphology and properties. Subsequent treatments such as annealing and cooling, electrical poling, and compression can be applied to further enhance the crystallinity of the b-phase of the PVDF and its copolymers. 97,[191][192][193]195 2.2.8.1 b-phase content during electrospinning. Electrospinning favours the formation of b-phase formation or induces an ato b-phase transformation due to its unique in situ electrical poling and mechanical stretching effects. The b-phase content in polymeric bres is dependent on the solvent type, additives and electrospinning parameters. For example, when electrospinning PVDF from DMF solution, a b-phase fraction of 75% and a crystallinity in the range of 49-58% can be produced. It is evident that solvents with a higher dipole moment produce PVDF lms with g-phase crystallinity, while solvents with lower dipole moment result in the nonpolar a-phase. The formation of the ferroelectric b-phase can be controlled by the composition of the solvent, notably the water content. 196 Using a hydrated salt in the casting solvent formed lms with a high bphase content. Table 2 summarises some typical results for effects of electrospinning parameters on the formation of the polar b-phase.
By adjusting the electrospinning conditions to narrow the bre diameter or increasing the electrospinning voltage, more b-phase was obtained, which is ascribed to the increased elongation and a larger electric eld that acts as an increased poling eld on the polymer jet. 197 When electrospinning PVDF from a N-methyl-2-pyrrolidinone/acetone (5/5 v/v) mixed solution at 16 wt% concentration, PVDF nanobers with 95% of b-phase were collected from a rotating drum at 800 rpm. 198 When electrospinning 20 wt% PVDF from DMF/acetone (6/4 v/v) solution 199 in the presence of 3 wt% of tetrabutylammonium chloride (TBAC), almost pure b-phase bres were produced. The TBAC salt is believed to facilitate the degree of hydrogen bonding between water molecules and the uorine atoms of the PVDF and hence induce more trans-conformation. In contrast, only the aand g-phase was detected in the spin-coated samples from the same solutions. It is believed that the TBAC additive induced local conformational changes and electrospinning promotes inter-chain interactions.
A higher fraction of polar b-phase can generally lead to higher d 33 piezoelectric coefficients, 34 and therefore a higher voltage output in energy harvesting applications, however the piezoelectric performance of PVDF bers is also affected by the total dipole moment, i.e., the orientation of dipoles that is closely related to the poling effect. 2.2.8.2 Orientation of molecular dipoles during electrospinning. Given the high applied electrostatic elds and polymer jet characteristics of the electrospinning process, it is believed that electrospinning can not only facilitate more ferroelectric phase formation, but also induce dipole orientation in the polymer bres. A number of studies have reported exible, high-output piezoelectric nanogenerators based on PVDF bers using near-eld electrospinning (NFES) or conventional far-eld electrospinning (FFES) processes. 200,201,[201][202][203] However, the effects of the electrospinning process on the dipolar orientation of the polymer bres are still not fully understood and con-icting results have been reported. 202,203 During electrospinning, a polymer solution experiences a number of forces. The rst is a shear force when it ows through a capillary needle at a high rate. The second is a coulombic force when the jet is elongated and accelerated by the applied electric eld. When a rotation disk collector is used to collect the bers, a mechanical force may also be applied. These three forces can cause polymer chains to be aligned and/ or stretched in the spinning direction. Using a rotating drum or disk as a bre collector can exert an additional mechanical stretching, and promotes the formation and alignment of the caxis of the b-phase crystallites along the ber axis. 199 One study 204 reported that rotation of the collecting drum has a pivotal role in the enhancement of b-phase formation and degree of the orientation of the bers in the electrospun mats. However, another study found that the degree of orientation and the polymorphism behaviour of the bres did not vary signicantly with either the rotating disk speed or the size of the spinneret used. 199 This implies that the formation of the bphase is likely to be caused by the coulombic force imposed by the electric eld rather than the mechanical and shear force exerted by the rotation disk collector and spinnerets.
The coulombic force may cause conformational changes to the straighter TT conformation, and hence promote the formation of b-phase. To seek evidence of dipole orientation during the electrospinning process, Sun et al. 198 compared electrospun PVDF nanobers with bres made by a force-spinning process (mechanical stretching without electrostatic force), in which the nanobers were produced by an electrostatic force and a centrifugal force. It was found that both brous mats formed showed aligned PVDF bres with a high bphase content of 95%. However, the force-spun PVDF bers did not show piezoelectricity, although it had a high b-phase content, since the mechanical force may only induce a phase transformation, but fails to align the ferroelectric dipoles. In comparison, the FFES process can lead to an electric poling eld ($1 kV cm À1 ) on the polymer jet, thus inducing polarization of the crystallites with a preferential orientation along the bres; see Fig. 20.
A study on PVDF/polar polyacrylonitrile and PVDF/nonpolar polysulfone nanobers 205 concluded that mechanical stretching is more effective than electric poling to induce the formation of ferroelectric phases. The b-phase was more stable in the polar polyacrylonitrile blends than in the nonpolar polysulfone/PVDF blends, which indicates that apart from the mechanical stretching, the local electric eld-dipole interactions determine the nucleation and growth of polar PVDF phases. This reects that electrical poling is more effective than mechanical stretching in enhancing the piezoelectric properties of PVDF nanobers. 205 2.2.8.3 Synergistic effects of nanoparticles and electrospinning. While the benets of nanocomposites have been discussed, the addition of nanoparticles has also been used in the electrospinning process. The possible mechanism of the formation of b-phase PVDF with the aid of templates with a large surface area is proposed in Fig. 21.
Both OMMT 149 or modied CNTs, 162,206 with their intrinsically large aspect ratio, are capable of interacting with the positive charged -CH 2 or negative -CF 2 groups of PVDF, resulting in the large scale conformation of polar phases, in particular the b-phase. Clay can increase and stabilise the polar phase in PVDF bres, 207 and act as a processing agent to facilitate electrospinning. 208 Particles with a smaller aspect ratio, such as Ag, metal salts or ionic liquid, also induce the formation of polar b-phases, but are not as effective as the particles with a high aspect ratio. Electrospun PVDF/room-temperature ionic liquid (RTIL) composite nanobers 206 based on 1-butyl-3-methylimidazolium hexauorophosphate [BMIM][PF6], exhibited almost 100% of b-phase, which differs from the dominant gphases observed for melt-blended PVDF/RTIL blends. 209 Two different nanoscale particles: carboxyl-MWCNTs and Ag-doped MWCNTs were used to modify the piezoelectric coefficient of PVDF electrospun bers. 188 The high voltage electrostatic eld generated extensional forces on the polymer chains that aligns dipoles. The Ag-CNTs lled PVDF electrospun bers exhibited the highest piezoelectric coefficient (d 33 ¼ 54 pm V À1 ) in contrast to PVDF/CNT bers (35 pm V À1 ) and pure PVDF (30 pm V À1 ). In another study, silver nanowires (AgNWs) doped PVDF bers were electrospun in a mixed DMF/acetone solution. 210 The b-phase content in the PVDF was increased by the presence of AgNWs, and the piezoelectric coefficient d 33 was 29.8 pC N À1 for the nanobers webs containing 1.5 wt% AgNWs, which is close to that of P(VDF-co-TrFE) (77/23).
Additives can aid the electrospinning process, and also improve the piezoelectric performance of the composites bers. The presence of inorganic salt LiCl of 0.00133 wt% in PVDF DMF/acetone (4/6) solution in 16 wt% concentration, 211 decreased the ber diameter from 340 nm to 65 nm due to the increased charge carried by the jet, and increased the b-phase formation to 93.6%. The voltage response of PVDF was increased from 1.89 V to 8 V for composite bers with 0.00133 wt% of LiCl, which is of interest for harvesting. Alternatively, increasing the collector drum speed up to 8.4 ms À1 lead to the alignment of b-crystallites along the ber axis without a significant effect on the formation of b-phase and output voltage. The addition of acetic acid signicantly reduced the density and size of the beads, while the addition of TBAC effectively removed all traces of beads from the electrospun bers. The b-phase enhancement induced by TBAC is likely to be caused by the hygroscopic nature of the salt, which retains water in the bers and leads to hydrogen bonding between the water molecules and the uorine atoms of PVDF. 196 Non-woven ber mats of PVDF/BaTiO 3 nanocomposites 212 with randomly oriented ber diameters ranging between 200 nm and 400 nm have been fabricated. The PVDF electrospun mats exhibited a mixed crystalline phase consisting of both aand b-phases. However, the addition of BaTiO 3 was found to result in an increase in the b-phase content in electrospun PVDF/BaTiO 3 mats. By increasing the bre alignment in the PVDF brous membranes, the piezoelectric response was enhanced. 213 For randomly orientated membranes with a BaTiO 3 concentration of 20 wt%, the output voltage was 0.1 V and corresponded to a 7 mm displacement at 1 Hz. 185 By uniaxial-alignment of the composite bres, the piezoelectric output voltage was increased with increasing BaTiO 3 concentration, and reached 0.48 V at 6 mm deection, which is 1.7 times higher than PVDF bers of 0.27 V when subjected to the same deformation, see Fig. 22. 214 The increased piezoelectric response of uniaxial-aligned BaTiO 3 -PVDF nanobers in comparison with randomly oriented PVDF bers and thin lms suggests their possible uses in energy harvesting and as power sources in miniaturized electronic devices such as wearable smart textiles and implantable biosensors.
2.2.8.4 Electrospinning technology. A variety of electrospinning methods have been developed in order to produce aligned or doped nanobers for energy applications. For example, PVDF nanobers were produced by a bubble electrospinning technique, where the production rate of the obtained PVDF nanobers was higher (13.93 mL h À1 ) than that of the reported traditional electrospinning (1-5 mL h À1 ). 222 The crystallinity of the PVDF nanobers was higher than in traditionally synthesized PVDF powders. PVDF bers were also investigated via non-uniform eld electrospinning where the air pressure and PET substrate thickness were varied to align the PVDF bers, and increase the level of piezoelectricity. 223 PVDF/Fe 3 O 4 nanocomposite bers were prepared under magnetic eld assisted electrospinning. 224 Two Helmholtz coils were mounted on the electrospinning apparatus to create a uniform magnetic eld. The PVDF in DMF/acetone (3/1) solution with a concentration of 18 wt% were mixed with Fe 3 O 4 nanoparticles in the size of 20-30 nm at PVDF ratios of 1 : 5, 1 : 10 and 1 : 15. The application of an electromagnetic eld during ber deposition resulted in improved orientation of the polymer ow towards the grounded electrode and led to smoother bers with diameters in the range of hundreds of nanometers. A magnetic eld response of the nanobers with higher magnetic elds was observed.
During the conventional electrospinning process, the fast evaporation of the solvent and the Coulomb forces generated among the induced charges inside the electried jet causes the polymer jet to follow a curved shape. 225 These cause the elec-tried jets to spin in a looping path towards the collector, and thus the bending instability results in randomly oriented nanobers. 226 Well-aligned nanobers are oen required for energy, sensor and biomedical applications, 221,227 but their production by electrospinning remains technically challenging. Different techniques have been developed to produce unidirectional aligned bers, such as modication of electrostatic eld, 228-230 mechanically wiring, and using a specially designed collector, including a fast rotating mandrel collector 221,231 or a parallel-electrode collector. 232 Centrifugal electrospinning 221 applies a centrifugal force and disperses a PVDF solution through a capillary, which causes elongation and thinning of the solution jet, and the ber is produced with no applied voltage. In a recent study, a hybrid centrifugal electrospinning process has been developed by integration of the concepts of the parallel-electrode electrospinning with centrifugal dispersion, that can produce highly aligned bers at a large scale, 221 see Fig. 23. Aligned PVDF bers were prepared across three-inch electrode gaps at an applied voltage of 15 kV and a spinneret rotation speed of 200 rpm from a 20 wt% PVDF solution with 3 wt% TBAC. Randomly oriented bers as well as aligned bers produced by the parallel electrodes were also prepared. All ber samples were 25 mm long with a cross-sectional area of 0.76 mm 2 . The samples were embedded in PDMS, connected via electrodes to a voltageoutput analyzer and clamped onto a nanomechanical tester to produce controlled strain rates, as shown in Fig. 23a. The aligned bers shown in Fig. 23b produced an output voltage of $3.04 mV at a strain of 0.10 compared to the only 0.059 mV for the randomly oriented bers. Although the individual PVDF bers in the randomly oriented specimen were likely to be poled, the randomly oriented poling directions in the membrane's bulk macrostructure resulted in a negligible response. A schematic of the centrifugal system is shown in Fig. 23f.
In a rotating collector conguration, the polymer bers are deposited and wrapped around a rotating mandrel. The degree of ber alignment mainly depends on the mandrel rotational speed. By simply mounting two polyimide lms onto a rotating cylindrical collector with a 1 mm gap from the collector surface, the air ow generated by the rotation of the collector can be redirected, and the electric eld at the gap between the lms can be altered; this is termed the 'end-point' control assembly method, 233 as shown in Fig. 24. The deposition of nanobers can therefore be controlled by the relative position of the gap and the collector. With this manufacturing setup, PVDF ber bundles were electrospun when operated at the tip-to-collector distance of 6 cm, external applied voltage of 8 kV, and the two polyimide lms separated by a distance of 5 mm. It was found that both the alignment and positioning controllability of the electrospun PVDF bers were improved as compared with conventional electrospinning with a rotating cylindrical collector.
The electrospinning process can be classied according to the spinning distance, for example, near-eld electrospinning (NFES, spinning distance < 1 cm), 'short-distance' electrospinning (spinning distance of 1-8 cm) 195,[234][235][236][237][238][239] and conventional electrospinning (spinning distance > 8 cm). The main characteristic of NFES is that the jet does not have a 'whipping' movement within the short spinning distance, and the macromolecular chains can align along the ber length. In the conventional electrospinning process, the polymer jet undergoes whipping and solidication before it deposits on the substrate. The electrospinning of PVDF at a short spinning distance range (1-8 cm) can form highly interconnected bers throughout the web. The mechanical properties are higher than bers made by conventional electrospinning due to the inter-ber connections stabilizing the brous structure. 195 Both NFES and short-distance electrospun PVDF nanobers could be more suitable for the development of robust energy harvesters.
In order to grow b-phase extended-chain crystallites in PVDF bers, a high electric eld (1.6 Â 10 7 V m À1 ) elongates the polymer jet and a strong extensional force is applied when the glass tube collector rotated at 700-2100 rpm. A further introduction of CNTs enhances the b-phase formation, and the benets of CNT additions have been discussed in Section 2.2.7.4. The PVDF bers exhibited a large deection under high electric eld. Bending resulted in a mechanical strain (0.05-0.1%) that was distributed along the PVDF bers, and the maximum voltage and current output of the piezoelectric energy harvesters were 43.6 mV pp and 240 nI pp at a 15 Hz impact frequency, respectively. A exible energy harvester was made by patterning of ordered PVDF/poly(g-methyl L-glutamate) (PMLG) composite bers produced by NFES onto a PET lm. The NFES process improved the piezoelectric properties of the PMLG/ PVDF composites, resulting in better orientation of their dipoles, a high ultimate stress (27.5 MPa), and a high Young's modulus (2.77 GPa). The energy harvester could capture ambient energy with a maximum peak voltage of 0.08 V, a power of 637 pW, and the energy conversion efficiency is 3.3%. The electro-mechanical energy conversion efficiency of this PVDF/ PMLG energy harvester was up to three times higher than those of pure PVDF and PMLG based energy harvesters. 237 The NFES and hollow cylindrical near-eld electrospinning (HCNFES) process was used to fabricate piezoelectric PVDF/MWCNT nanobers. 240 When comparing NFES and HCNFES, the HCNFES process applies a high electric eld with an in situ mechanical stretching for alignment of dipoles along the longitudinal direction of PVDF nanober. Therefore, the PVDF nanobers fabricated using HCNFES can be of smaller diameter and higher b-phase content with higher piezoelectric activity for harvesting.
Examples of structuring ferroelectric polymers in devices
This section describes some of the potential harvesting devices that exploit the properties of ferroelectric properties that have been optimised using the methods described above. The emphasis of this section is not on the design of the devices but to provide examples on how the optimisation of the ferroelectric properties of the polymers using the processes described in this review can lead to improved performance. Exploitation of the ferroelectric materials will be achieved by a combination of both harvester device design and optimisation of the structure of the piezoelectric material at a range of scales (molecular-, micro-and macro-structure). Since all pyroelectrics are piezoelectric, researchers have attempted to combine both pyroelectric and piezoelectric harvesting approaches. Table 3 compares the relevant equations for a pyroelectric subjected to a temperature change (DT) and piezoelectric subjected to a stress (Ds) with similarities in the relationships between current, voltage and stored energy. Due to their similarities there is interest in hybrid piezoelectric-pyroelectric harvesting systems 241-244 whereby a combination of temperature change and stress is applied. In such systems care must be taken to ensure the changes in polarisation are constructive and enhance the power generation of the harvesting device. 241 As discussed by Sebald et al., the frequencies of temperature and vibration can be different and there is a need to optimise the electronics for such a hybrid system. 245 Flexible and stretchable piezo-and pyro-electric generators have attracted considerable attention in energy harvesting systems, especially in wearable and portable electronics, electronic skins, touch screens and electronic textiles. The challenges of exible and stretchable nanogenerators include the limited output power generation, low efficiency at low frequency, mechanical weakness, low stability and durability. The exibility and stretchability of the devices rely on the intrinsic mechanical properties of polymers and elastomers, or they can be realised by intelligent design of the device structures. 246 For instance, through the fabrication of foam-like structures, micro-patterning, 243 arch-shape assembly, electrospun membranes and textiles. The use of structuring technologies expands the range of potential materials from elastomer to semi-crystalline polymers, and hybrid nanocomposites. Nanostructuring can also enhance output power density by integration of piezo-and pyro-electric generators with other harvesting mechanisms, such as the triboelectric effect, and energy storage devices.
Curved and multi-layered structured devices
A stretchable hybrid piezo-and pyro-electric energy harvester was fabricated by using micro-patterned PDMS/CNTs composite as an electrode, which also makes the nanogenerator stretchable and exible. Graphene was used as a top electrode for improved heat transfer, and a P(VDF-co-TrFE) copolymer layer provided piezo-and pyro-electric performance, as described in Section 2.2.1. Under the effects of a thermal gradient and mechanical strain from 0 to 30%, the pyroelectric output voltage was stable at 0.4 V. 243 The potential of the material to harvest both mechanical loads (s) and temperature changes (DT) was examined. The total change in polarisation is expressed as: A multilayered exible PVDF curved generator was studied by investigating the effects of using a curved structure and tailoring the substrate material. 246 By stretching the device to a 1 cm displacement in the x-direction, the PVDF lm, without a polyimide substrate, generated a low output voltage of only 0.1 V, while with a substrate it generated over 100 V. In addition, PVDF with a curved structure generated 86 V, in comparison to a at PVDF lm that only produced 15.7 V. As a result, the attachment to a substrate to a curved structure allowed the PVDF to be subjected to a higher external force, and induces a higher stress and produces a higher output power. The multilayered curved nanogenerator produced a peak output voltage of $200 V and a peak output current of $2.7 mA. The output power density of the generator reached $17 mW cm À2 , which could illuminate over 950 LED bulbs. This concept was reported to provide the groundwork to enhancing the output power of conventional piezoelectric generators, thereby enabling novel approaches to realizing self-powered systems.
A hybrid piezoelectric/triboelectric generator 247 was fabricated by vertically-stacking two functional layers, as shown in Fig. 25. An arch-shaped piezoelectric generator with a Au/PVDF/ Au structure on the top, and a touch-and-release type triboelectric generator with a PTFE/Al structure on the bottom. In the Table 3 Comparison of relevant equations for pyroelectric p ¼ dP s /dT (C m À2 K À1 ) and piezoelectric systems d ij ¼ dP s /ds (C N À1 or C m À2 /N m À2 ) and 3 T 33 is permittivity at constant stress 16
Parameters
Pyroelectric Piezoelectric
33
 h  Ds triboelectric generator, electrical charges are generated due to the charge transfer between two thin organic/inorganic lms that exhibit a different surface electron affinity. The arch-shaped piezoelectric generator produced electricity by the d 31 mode. The device had a common shared electrode as a bottom electrode of the piezoelectric generator and as a driving electrode of the triboelectric generator. With two full-wave bridge diodes connected to the hybrid generator, the piezoelectric and triboelectric outputs were effectively combined. The hybrid open-circuit output voltage and current density were approximately 370 V and 12 mA cm À2 , respectively, and the output power density was $4.44 mW cm À2 . The hybrid nanogenerator produced a high output power even at a mechanical force of as small as 0.2 N, enough to light 600 LEDs.
Nanoconnement based devices
P(VDF-co-TrFE) nanowires with a diameter of approximately 196 nm were prepared by using an AAO template-wetting method, 248 with the nanoconnement promoting high crystallinity and preferential molecular dipole orientation in the polymer nanowires; as discussed in Section 2.2.4. The as-prepared selfpoled polymer nanowire exhibited a peak electrical output voltage of 3 V and peak output current of 5.5 nA when subjected to a strain rate of 0.1% s À1 , which is comparable to those reported highest efficiency values estimated from electrospun nanowire generators. 234 The energy conversion efficiency was approximately 11%.
Foam-like structured devices
Meso-and micro-porous structures are important for tailoring the mechanical energy harvesting performance of polymer lms. 249 Porous PVDF nanogenerators have been fabricated by templating or electrospinning methods (Section 4) to further enhance the piezoelectric properties while providing the materials with stretchability and exibility. In addition to tailoring the crystalline structures of PVDF-based materials, the pore size and porosity of the materials can also affect the properties. By using a templating method, mesoporous PVDF thin lms were prepared by casting PVDF/ZnO nanoparticles solutions, following by etching of the ZnO nanoparticles, as shown in Fig. 26A. The benets of nanocomposites and electrospinning nanocomposites have been discussed in Section 2.2.7 and 2.2.8, respectively. The pore size and porosity of the lms were controlled by the size and volume fraction of the ZnO particles. 249 With a ZnO particle size of 35-45 nm, the output voltage increased from 3.5 to 11 V as the porosity increased from 6.5 to 32.6% when the ZnO mass fraction increased from 10 to 50%, but the voltage decreased to 8.3 V as the porosity further increased to 45.5% at a ZnO mass fraction of 70 wt%. The mixing of ZnO with PVDF helped to from a higher fraction of bphase in the PVDF, and reached a maximum fraction of b-phase at a ZnO content of 50 wt%, where the output voltage and current of the porous PVDF lm nanogenerator 28 mm thick was 11 V and 9.8 mA at a frequency of 40 Hz, respectively. Porous PVDF lms were fabricated with a ZnO nanowire array template-assisted preparation method, as shown in Fig. 26B. 250 With a sonic power of 100 dB at 100 Hz, the porous PVDF with a pore size of 2 mm showed an enhancement in the peak-to-peak piezoelectric potential (open circuit voltage)/ piezoelectric current (short circuit current) with an output of 2.6 V/0.6 mA, which is over 5.2 times (piezoelectric potential) and six times (piezoelectric current) higher compared to that of the bulk lm (0.5 V/0.1 mA). The device could produce a rectied power density of 0.17 mW cm À3 . For the porous PVDF lms with pores of 700-900 nm and 2.7 mm in thickness, an output voltage 2.84 V and an electric eld of 1.05 Â 10 6 V m À1 were measured, which is ascribed to the geometrical strain connement effect, as shown in Fig. 26C. 251 The pore structure can reduce the strain on the orthogonal direction, to allow the development of strain that is conned to the axial direction. The porous structured nanogenerators showed a higher performance compared to ber-like generators, such as a single PVDF nanober (30 mV/3 nA, 6.5 mm diameter/600 mm length), 234 PVDF nanobers (20 mV/0.3 nA, sample thickness and area were not indicated), 202 PVDF mats (z1 V, 100 mm thickness/1 cm 2 area, output current not indicated), 252 and PVDF nanober membranes (z2.2 V/z4.5 mA, 140 mm thickness/2 cm 2 area). 203 Porous P(VDF-co-TrFE) copolymer lms have also been prepared via an immersion precipitation and phase inversion method, 253 where the porosity can be varied by changing the type of solvent and nonsolvent. By using 15 wt% of P(VDF-co-TrFE) (60/40) copolymer in MEK with ethanol as a nonsolvent, a porous lm with an average pore size of 2.2 mm and 33% porosity was produced. As shown in Fig. 26D, the porous lm had a coercive eld, E c of 30.5 MV m À1 , similar to those of commercial copolymer thin lm, but the relative permittivity 3 0 was 39.9 which is higher than commercially available thin lm material (3 0 $ 28.8). The pyroelectric coefficient of commercial thin lm and porous lms were 4.5 Â 10 À5 and 4.3 Â 10 À5 C m À2 K À1 at room temperature, respectively, and both exhibited peak values of 1.4 Â 10 À4 and 1.2 Â 10 À4 C m À2 K À1 at 50 C, respectively, which is the transition temperature from the ferroelectric b-phase to the paraelectric a-phase phase. In this case, the porous structure improved the thermal response of the lms, but reduced the pyroelectric coefficient. Therefore, porous structure and porosity are a key features to tune the properties of the materials for harvesting applications.
Fibrous structured devices
As mentioned for electrospinning (Section 2.2.8), the NFES technique can produce in situ poled and well-aligned nanobers in a continuous manner due to the strong electric elds (>10 7 V m À1 ) 254 and stretching forces during the electrospinning process. It can realize direct-write and integrated PVDF nanogenerators with high energy conversion efficiency. 234,255 The piezoelectric coefficient d 33 is À63.25 pm V À1 for a single NFES bre, 238 or À57.6 pm V À1 for PVDF brous mats, 256 which is twice as large than that reported for PVDF thin-lms (about À15 pm V À1 ). The typical electrical outputs of the direct-write PVDF nanogenerators are 5-30 mV and 0.5-3 nA. 215 In comparision, randomly distributed PVDF nanobers prepared by conventional electrospinning process did not show a measurable electrical output. A number of serially integrated layers of the fabricated nanober were encapsulated in a device consisting of Fig. 26 Images of (A) mesoporous PVDF thin film templated with ZnO nanoparticles, and the voltage output of a PVDF thin film NG (fabricated from a 50% ZnO mass fraction mixture) generated during one cycle of surface oscillation. The blue and red curves were collected under forward and reverse connections, respectively; 249 (B) cross section SEM images. Scale bars are 5 mm. A porous PVDF nanostructure using a ZnO nanowire template, and the piezoelectric potential (blue) and piezoelectric currents (red) from the porous PVDF and bulk structure under the same force; adapted with permission from ref. 250 a PDMS polymer on a PVC substrate, and an increase of output voltage was obtained due to the increasing number of layers via a summing up the external piezoelectric potential. A nanogenerator that comprised 20 000 rows of well-aligned PVDF NFs was able to create a peak output voltage of $4 V and a current of 75 nA. A three-layer integrated nanogenerator could reach a maximum output voltage and current up to 20 V and 390 nA, respectively. By attaching the nanober-based device on the human nger under a folding-releasing cycle at $45 , the output voltage and current could reach 0.8 V and 30 nA, respectively, making the nanober based devices promising for exible and wearable electronics. 238 Fig . 27a shows a hollow cylindrical near-eld electrospinning setup. 215 Large arrays of PVDF bers were produced with a high concentration of b-phase. The bers were deposited on a PET substrate and xed with copper foil electrodes at both ends to form a exible PVDF energy harvester, as shown in Fig. 27b. At a strain of 0.05%, a maximum peak voltage and current was measured to be 76 mV and 39 mA, respectively, as shown in Fig. 27c. 215 The co-doping of Ce 3+ and graphene to PVDF electrospun nanobers helped to align and achieve in situ poling of the polar ferroelectric crystal phases. 220 The sensitivity and energy harvesting performance of the composite bres were tested for different applications. As an ultrasensitive pressure sensor, it could detect a pressure as low as 2 Pa. As an acoustic nanogenerator, it generated an output voltage of 11 V and 6.8 mW of maximum power by the application of a 6.6 kPa of pressure amplitude, which could light up 10 blue light emitting diodes (LEDs). For sound-driven power generation, when a sound intensity of $88 dB was applied to the nanogenerator, an AC output voltage of approximately 3 V was obtained but no current was reported; however the device could power three blue LEDs. These properties are ascribed to the synergistic effects of graphene, Ce 3+ doping and electrospinning. The electrospun nanogenerator was reported to be promising for application as a pressure sensor, mechanical energy harvester, and effective power source for portable electronic and wearable devices.
Single PVDF nanober prepared by NFES only show an electrical output in tens of millivolts under bending. 234 With a metallic coaxial needle injector, hollow PVDF ber tubes were produced by NFES. Near-eld electrospinning was shown to enhance the energy harvesting performance of hollow PVDF piezoelectric bers. 235 The elongation of the bre tubes was greater than that of solid PVDF bers, with a tensile strength of 32.5 MPa. The output voltage of the ber tubes was 71.7 mV with an external load resistance of 6 MU; the output power was also signicantly greater (856 pW) than the solid PVDF ber which has output voltage of 45.7 mV and the maximum output power of 347 pW. As a result, the power generation of the PVDF ber tubes, while small in magnitude, was 2.46 times higher than that of the solid bers. With a rotating glass tube collector, highly aligned PVDF nanobers were produced by the NFES process. 236 All-bre piezoelectric fabrics nanogenerators 257 were fabricated by assembly of knitted high b-phase ($80%) piezoelectric PVDF monolaments as the spacer yarn interconnected between silver coated polyamide multilament yarn layers acting as the top and bottom electrodes. With an effective area of 15 cm  5.3 cm, the 3D spacer piezoelectric fabrics exhibited an output power density in the range of 1.10-5.10 mW cm À2 at applied impact pressures of 0.02-0.10 MPa. This was reported to be higher than 2D woven and nonwoven piezoelectric structures. The piezoelectric fabrics can also be coupled with the knitted and screen printed supercapacitors for textile based energy harvesting storage in wearable electronics. 258 Clearly, these harvesting devices demonstrate that the optimisation of PVDF at multiple scales can improve the performance of energy harvesting systems.
Copolymer and nanocomposite based devices
Copolymers and nanocomposite materials have been used to create energy harvesting devices. Bhavanasi et al. reported enhanced piezoelectric energy harvesting performance in bilayer lms of poled PVDF-TrFE and graphene oxide (GO). Bilayer lms of poled P(VDF-co-TrFE) and GO 259 exhibited a voltage output of 4 V and power output of 4.41 mW cm À2 for energy harvesting as compared to poled P(VDF-co-TrFE) lms alone that had a voltage output of 1.9 V and power output of 1.77 mW cm À2 . The enhanced voltage and power output in the presence of the GO lm was thought to be due to the combined effect of electrostatic contribution from the GO, residual tensile stress, enhanced Young's modulus of the bilayer lms, and the presence of space charge at the interface of the P(VDF-co-TrFE) and GO lms. The higher Young's modulus and relative permittivity of GO was thought to facilitate efficient transfer of mechanical and electrical energy. Zhao et al. 260 produced piezoelectric nanocomposites composed of barium titanate nanoparticles with a PVDF matrix. By a simple and solvent evaporation process, the BaTiO 3 nanoparticles and PVDF composites where formed with an oriented structure and the highest open-circuit voltage reached 150 V. Siddiqui et al. 261 produced lead free and exible piezoelectric nanogenerators based on a piezoelectric nanocomposite thin lm of barium titanate nanoparticles embedded in a highly crystalline polyvinylidene P(VDF-TrFE) polymer for charge storage, Fig. 28. The nanocomposite with up to 40 wt% of BaTiO 3 in the nanocomposite produced an output voltage of 9.8 V and output power density of 13.5 mW cm À2 under cyclic bending, comparable to the output of conventional inorganic polycrystalline lead zirconate titanate devices. The high performance of nanocomposite system was thought to be attributed to the high effective piezoelectricity of the crystalline P(VDF-TrFE) and BaTiO 3 .
Summary and perspective
This review has provided a detailed examination of the use of exible PVDF based ferroelectrics for energy harvesting applications. The potential mechanisms by which a ferroelectric can harvest vibrations or thermal uctuations has been explored. In terms of improving the ferroelectric properties for harvesting applications, a wide variety of properties of interest can be tailored. This can include the piezoelectric coefficients (such as d 33 and d 31 ), relative permittivity, pyroelectric coefficient, Curie temperature (T c ), the electric eld required to activate and align dipoles in ferroelectric domains (coercive eld, E c ) and the remnant polarization (P r ) which is related to the number of dipoles "locked" in aer the poling process. The main ferroelectric phase of PVDF is the b-phase and approaches to maximise the fraction of the ferroelectric phase to optimise the ferroelectric, piezoelectric and pyroelectric properties of polymer materials are described in detail; these include the inuence of polymer processing conditions, co-polymerisation, heat treatment, nanoconnement, blending, porosity, ionic liquids, forming nanocomposites that contain dielectric or conductive llers and electrospinning. A wide variety of processing conditions and methods are available to optimise the properties above for the desired application; a combination of these methods can also be exploited. While a number of these approaches have been used to design and tailor exible material for harvesting applications, such as forming nanocomposites and electrospinning, some areas have yet to be explored, such as the use of nanoclays and ionic liquids. The majority of work concentrates on processing and experimental characterisation and opportunities exist for modelling these complex materials are a range of scales to inform the experimental efforts. The range of interactions and potential benets are highlighted in Fig. 29. We have seen that concept of harvesting typically covers systems at relatively lower power levels (nW to mW) for applications such as low power electronics or for wireless sensors and reduce reliance on batteries, electric cables and provide autonomous sensors or devices. Less effort has considered the use of Olsen types cycle for higher power/larger thermal harvesting applications and are worthy of investigation.
Applications include exible and stretchable piezo-and pyroelectric generators have attracted considerable attention in energy harvesting systems, especially in wearable and portable electronics, electronic skin, medical devices touch screen and electronic textile with stretchable properties. Since ferroelectric polymers are both piezoelectric and pyroelectric properties, there is the prospect to harvest energy from multiple sources including vibration and thermal uctuations so that the design of hybrid systems is possible. This can also be combined with additional 'non-ferroelectric' based harvesting mechanisms in polymers, such as tribo-electric effects. Ferroelectric polymers, such as PVDF and its copolymers can also be used as energy storage materials due to their high dielectric strength, permittivity and relaxor properties; therefore there is also scope for combining harvesting and storage applications, such as multilayer structures and devices. While ceramic based materials with high Curie temperature and high ferroelectric activity are available the ability of polymers to be processed at lowtemperatures, their low density, bio-compatibility, low stiffness, exibility and mechanical robustness, such as toughness and high strains to failure will ensure there remains a number of applications where these materials are of continued interest for harvesting applications. | 2018-04-30T17:18:10.852Z | 2017-02-14T00:00:00.000 | {
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119243282 | pes2o/s2orc | v3-fos-license | Laser Driving Highly Collimated $\gamma$-ray Pulses for the Generation of $\mu^-\mu^+$ and $e^-e^+$ Pairs in $\gamma-\gamma$ Collider
A scheme to generate highly collimated $\gamma$-ray pulse is proposed for the production of muon and electron pairs in $\gamma-\gamma$ collider. The $\gamma$-ray pulse, with high conversion efficiency, can be produced as the result of electron phase-locked acceleration in longitudinal electric field through the interaction between an ultra-intense laser pulse and a narrow tube target. Numerical simulation shows that 18\% energy of a 10-PW laser pulse is transferred into the forward $\gamma$-rays in a divergence angle less than $ 3^\circ$. The $\gamma$-ray pulse is applied in $\gamma-\gamma$ collider, in which muon pairs can be produced and electron pairs can be enhanced by more than 3 orders of magnitude. This scheme, which could be realized with the coming 10PW class laser pulses, would allow the observation of a $\gamma-\gamma$ collider for electron and muon pairs in laboratory.
The laser intensity above 10 23 W/cm 2 , which will be accessible by the under construction laser facilities [1,2], at which a large portion of the laser pulse energy can be transferred into γ-rays [3][4][5] in the laser matter interaction at the intensity. The γ-ray source with high conversion efficiency has attracted particular interest due to its wide applications [1,[6][7][8][9][10][11][12][13], among which the research on quantum electrodynamics (QED) should be one of the most important. In the interaction, although the laser field strength is several orders of magnitude lower than the Schwinger field E crit = 1.3 × 10 18 Vm −1 [14], QED effects [6,[15][16][17] become significant.
According to QED, matters can be created from light through Breit Wheeler (BW) [6,7,[18][19][20] process. In the linear BW process, only two photons involved in one collision, e − e + pairs could be generated through γ-photon [8] or γ −γ [9], and the next challenges should be the generations of much heavier leptons such as muon and tau pairs. The 10-PW laser pulse can produce a huge number of γ-rays [5,6,21,22] whose energy are much higher than the rest mass of muon. It is possible to generate muon pairs in the γ − γ collider driving by 10-PW laser pulses. However, µ − µ + pair generation in γ −γ collider has never been proposed due to the large divergence angle (∼ 30 • ) of the γ-rays [5,6,21,22]. Although efforts had been made to enhance the collimation of γ-rays [23,24], there is no distinguished enhancement on the collimation.
In this Letter, highly collimated γ-ray pulse is generated in the interaction between a 10-PW laser pulse and a narrow tube target. The electron collimation can be improved by phase-locked acceleration of longitudi-nal electric field E x . The γ-rays are generated near the tube inner boundary where the electrons are wiggled by the space charge field E ys and self-generated field B zs . Particle-in-Cell (PIC) simulation shows that the forward γ-rays which occupy 18% of the laser pulse energy are collimated into a divergence angle less than 3 • . The brilliance could be two orders of magnitude higher than the previous studies [4][5][6][21][22][23][24]. A γ − γ collider, which could produce muon pairs, is proposed using the γ-ray pulses, in which the production of electron pair can be enhanced by 3 orders of magnitude than before [8,9]. Hence, the γ-ray pulses will allow the observation of the creation of µ − µ + and e − e + pairs from pure light.
In the narrow tube target, the electron dynamics, which impacts the γ-ray generation, is affected by the traveling longitudinal electric field [25] generated in the confined space [26]. However, the underlying physics of electron dynamics and radiation in the narrow tube target is never clearly described although it has been intense applied [26][27][28][29][30]. For simplicity, the electron dynamics in a plane wave The electron motion equations can be written as dp where v y = dy/dt, v x = dx/dt and γ = 1 + p 2 x + p 2 y . The vector potential is a y = a 0 cos(φ) and E y = −∂a y /∂t, B z = ∂a y /∂x. Here E, B, t, x, p and a are normalized by m e ω l c/e, m e ω l /e, ω l , ω l /c, m e c and m e c/e, ω l is laser frequency, c is the light speed in vacuum and m e is the electron mass at rest. The electron, initially at rest, is pulled into the laser fields at initial phase φ 0 . Then, d( (1) Equation (1) implies that p x → ∞ and dφ/dt → 0, From Eq. (3) and (4), the locking phase reads where σ is a small quantity and n = 0, ±1, ±2, · · · . Figure 1(a) gives the electron longitudinal momenta at t = 40π for different a 0 and φ 0 by assuming ρ = 0.39. Electrons with φ 0 ∼ (2n + 1)π are accelerated to high energy but small transverse momenta as σ ∼ 0. The maximum longitudinal momenta are proportional to a 0 . The lines with σ = ±0.1 are approximative the boundaries of the phase-locked acceleration phases. For a 0 > 200 here, most of the electrons can experience phase-locked acceleration whose phase ranges from φ 1 ≈ (2n + 1/2)π to φ 2 ≈ (2n + 3/2)π as shown in Fig. 1(a).
To model the generation of γ-rays, two-dimensional PIC simulations are performed with EPOCH [35][36][37]. A 10-PW p-polarized laser pulse with Gaussian spatial and sin 2 temporal profiles, duration of 30 fs and focal spot diameter of 2.0 µm at FWHM, irradiates at a gold narrow tube from the left side. The laser intensity 3.2 × 10 23 W/cm 2 , corresponding to a 0 ≈ 480, would be available in ELI [1]. The tube e − / Au +69 density is set to 276n c / 4n c , where n c is the critical density. The tube wall, whose thickness is 400 nm and inner diameter is 4 µm, is located 2-270 µm. The simulation box is 271 µm in longitudinal direction (x) and 10 µm in transverse direction (y). 5 macro-ions and 100 macro-electrons are initialized in each cell whose size is dx = dy = 5 nm.
Electrons are pulled into the narrow space and pushed into the tube wall by E yL . High density (∼ 500n c ) electron bunches, and E ys and E xs are generated as shown in Fig. 1(b & c) and Fig. 1(d & e). The transport of the electron bunches results in the generation of B zs [23] as shown in Fig. 1(h). The laser fields and the secondary fields (E ys , E xs and B zs ) compose the fields shown in Fig. 1. Comparing the locations of the electron bunches, E y , E x , φ 1 and φ 2 marked in Fig. 1(c), results demonstrate that almost all the electrons dragged into the narrow space can be locked into the acceleration phase of E x , which is consistent with the theory.
In the narrow space, the dragged electrons can be classified into three groups as shown in Fig. 1(c & g). The transverse force F ⊥ exerted on the electrons is where E y = E ys + E yL and B z = B zs + B zL .
In the first group, E yL ≫ E ys and B zL ≫ B zs , the electron dynamics can be described by our theory as the laser fields dominate the movement. Due to initial large v y as shown in Fig. 1(g), the electrons will cross the laser axis and move toward the other side of the tube where most of them could be trapped again by E xL . Hence, the electrons in the first group could be trapped and accelerated twice. In the second group, B zs could cancel the local space charge field, E ys ≈ cB zs . E y ≈ cB z can be still satisfied as shown in 1(e & h). As F ⊥v → 0, the electrons can be trapped by E x for a long time which follows our theory as well. After crossing the phase of E y , the electrons will be pulled back to the tube wall by F ⊥v .
In the third group, the electrons which are exposed to larger E ys than E yL as shown in Fig. 1(f) will be easily pulled back to the tube wall by E ys . Thus, the electrons in this group can only contribute to low energy radiation due to shorter acceleration time. The above discussions show that the electrons in the first two groups can be phase-locked accelerated by E x for a long time. Figure 2(a & b) show the fields exerting on the test electrons in the first two groups, and the electron which can be accelerated to several GeV in a distance < 100µm as shown in Fig. 2(c & d) is mostly contributed from E x instead of E y and B z . The energy of the emitted γ-ray photon [36,37] could be estimated as hν ≈ 0.44ηγm e c 2 , where η ≈ γ|F ⊥ |/(eE crit ). For the generation of γ-rays, the emitting electrons should be accelerated to high energy before being wiggled. In the tube wall, the direction of E y turns reversal as circled in Fig. 1(e), the transverse force F ⊥ becomes Hence, F ⊥w is much larger than F ⊥v . The electrons will be pulled back to the tube wall, where the larger F ⊥w will significantly enhance the production of γ-rays [38] as shown in Fig. 2(c & d) and Fig. 3(a). All the above characters indicate that the electron movement and acceleration, together with the acceleration in two stages marked in Fig. 2(c), well follow the above discussion. At 900 fs in the simulation, ∼ 90% of the laser energy is depleted and the energy conversion η γ from the laser to the forward γ-rays which occupy more than 99% of the γ-rays is ∼ 18%. Most of the γ-rays are generated near the tube inner boundary as shown in Fig. 3(a). The photons are highly collimated to θ γ ∼ 3 • as shown in Fig. 3(b). The γ-ray pulse duration is about 21 fs (6.3 µm) and the focal spot diameter is about 25 µm at FWHM as shown in Fig. 3(c). The average photon brilliance, as shown in table I, is much higher than the reported results [6,21,23,24]. It is also found that η γ , brilliance and the collimation increase with I, and 31% of the laser pulse energy is transferred into γ-ray pulse collimated into 2 • at I = 1.28 × 10 24 W/cm 2 (40 PW laser pulse).
The full 3D condition is simulated by reducing the target length to 50 µm, in which the results show same characters as 2D. In the experiment, one can use much thicker tube but same diameter as the thickness does not affect the result. In addition, technical methods are needed to minimize the influence of pre-pulse and to inject the laser pulse into the narrow space.
For the highly collimated γ-ray pulses, one of the most important application is γ − γ collider. Figure 4 shows the setup of γ − γ collider, in which the γ-ray pulses are generated from laser driving narrow tube targets. The γ-ray pulses collide in a angle of θ c which should fulfill the relation θ c < 180 • − 2θ γ to make sure that most of the µ − µ + and e − e + pairs are generated in the forward direction where a detector is placed. d, the length of the targets, is fixed to be 200 µm as almost all the photons are generated in 70-200 µm as shown in Fig. 3(a). r = 2 µm is radius of the targets. A distance d 1 from target end to the collision point is considered to minimize the e − e + pairs produced from multi-photon BW process [6]. As the laser intensity decreases significantly after the transporting in vacuum for a distance of d 1 . According to the Eq (6) in the ref. [9], the pair number from BW process can be estimated by where N γ1 and N γ2 are the photon number in the γ-ray pulses. The distance from the source location to the collision point is about 200µm. The size of collision area S c , which should be doubled for circular-polarized laser pulse, can be expressed as S c = π(d sin θ γ / sin(0.5θ c + θ γ ) + (2r cos θ γ + d sin θ γ )/ sin(0.5θ c − θ γ )) 2 /8. The photon-photon cross section σ γ1γ2 [19] is where r c is the classical muon (or electron) radius, β = (1 − 1/s) 1 2 , s = ε γ1 ε γ2 (1 − cos θ c )/2m 2 l c 4 , ε γ1 and ε γ2 are the photon energies, m l is the rest mass of the leptons. s > 1 is the threshold condition for pair generation.
To solve Eq (8) and (9), macro-photons which are produced from the PIC simulation are used to reduce the data size. All the photons are assumed to collide in θ c which is acceptable for highly collimated γ-ray here. The photon spectra is divided into 500 segments in the step of log 10 (ε γ (max))/500, and the photons in the same segment are assumed to collide with the other pulse in the same condition.
For the generation of µ − µ + pairs, r c = e 2 /m µ c 2 , where m µ is muon mass at rest. From Eq (8) and (9), it is found that the muon number is about 1.6 per shot from headon collision in the case of 10 PW laser pulse, while can be enhanced to 160 by using 40 PW laser pulses. For e − e + pairs production, r c is changed to e 2 /m e c 2 , where e is elementary charge. Through the numerical solution of Eq (8) and (9), 3.4 × 10 8 (2.8 × 10 10 ) pairs can be generated from head-on (θ c = 180 • ) collision with the γray pulses generated from 10 PW (40 PW) laser pulses. The average photon brilliance Br (photons/s/mm 2 /mrad 2 /0.1%BW) at 270µm away from the irradiating point, the photon number (> 100keV) Nγ in the γ-ray pulses, the divergence angle θγ of the γ-ray pulses, the pair numbers of e − e + and µ − µ + generated from the γ − γ collider driven by different laser pulse and collision in different angle θc. The pair number is 3 orders of magnitude higher than the reported results [8,9]. The effects of θ c is explored to see the robustness of this method. It is found that the pair number is significantly affected by θ c as shown in table I. To observe strong signal by the detector, optimal value of θ c should be larger than 90 • and less than 180 • − 2θ γ . Meanwhile, the pairs generated from trident process [39], whose optimal thickness is much thicker than the tube wall, can be ignored. Hence, the highly collimated γ-ray pulses would allow the observation of muon and electron pairs generation in γ − γ collider in laboratory.
10
In conclusion, the electron phase-locked acceleration of longitudinal field is illustrated theoretically to resolve the dynamics of energetic electrons in the interaction between an ultra-intense laser pulse and a narrow tube target. Energetic electrons are accelerated efficiently by longitudinal electric field and radiate strongly near the inner boundary of the tube, resulting in the generation of a highly collimated γ-ray pulse. The brilliance of the forward γ-ray pulse, from PIC simulation of 10-PW laser pulse, is two orders of magnitude higher than the reported results due to high conversion efficiency up to 18% and small divergence angle less than 3 • . The γ − γ collider, in which the µ − µ + pairs can be created from pure light and production of e − e + pairs can be enhanced by 3 orders of magnitude, is proposed by using the γ-ray pulses. Such highly collimated γ-ray pulse which could be realized with coming 10PW laser pulses, would benefit the research on quantum electrodynamics, the production of isotopes and laboratory astrophysics.
This work has been supported by the National Basic | 2017-12-24T06:07:00.000Z | 2017-12-24T00:00:00.000 | {
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54184027 | pes2o/s2orc | v3-fos-license | Screening effects on the excitonic instability in graphene
We investigate the excitonic instability in the theory of Dirac fermions in graphene with long-range Coulomb interaction. We analyze the electron-hole vertex relevant for exciton condensation in the ladder approximation, showing that it blows up at a critical value of the interaction strength \alpha = e^2/4\pi v_F sensitive to further many-body corrections. Under static screening of the interaction, we find that taking into account electron self-energy corrections increases the critical coupling to \alpha_c \approx 2.09, for a number N = 4 of two-component Dirac fermions. We show that the dynamical screening of the interaction has however the opposite effect of enhancing the instability, which turns out to develop then at \alpha_c \approx 0.99 for N = 4, bringing the question of whether that critical value can be reached by the effective coupling in real graphene samples at the low-energy scales of the exciton condensation.
We investigate the excitonic instability in the theory of Dirac fermions in graphene with longrange Coulomb interaction. We analyze the electron-hole vertex relevant for exciton condensation in the ladder approximation, showing that it blows up at a critical value of the interaction strength α = e 2 /4πvF sensitive to further many-body corrections. Under static screening of the interaction, we find that taking into account electron self-energy corrections increases the critical coupling to αc ≈ 2.09, for a number N = 4 of two-component Dirac fermions. We show that the dynamical screening of the interaction has however the opposite effect of enhancing the instability, which turns out to develop then at αc ≈ 0.99 for N = 4, bringing the question of whether that critical value can be reached by the effective coupling in real graphene samples at the low-energy scales of the exciton condensation.
Introduction.-The discovery of graphene, the material made of a one-atom-thick carbon layer, has attracted a lot attention as it provides the realization of a system where the electrons have conical valence and conduction bands, therefore behaving at low energies as massless Dirac fermions [1][2][3]. This offers the possibility of employing the new material as a test ground of fundamental concepts in theoretical physics, since the interacting electron system represents a variant of strongly coupled quantum electrodynamics (QED) affording quite unusual effects [4][5][6][7].
A remarkable feature of such a theory is that a sufficiently strong Coulomb interaction may open a gap in the electronic spectrum. This effect, already known from the study of QED [8], was first addressed in graphene in the context of the theory with a large number N of fermion flavors [9][10][11][12]. The existence of a critical point for exciton condensation was also suggested from secondorder calculations of electron self-energy corrections [13]. More recently, Monte Carlo simulations of the field theory have been carried out on the graphene lattice [14,15], showing that the chiral symmetry of the massless theory can be broken at the physical value N = 4 above a critical interaction strength α c ≈ 1.08 [14]. The possibility of exciton condensation has been also studied in the ladder approximation [16][17][18][19], leading in the case of static screening of the interaction to an estimate of the critical coupling α c ≈ 1.62 for N = 4 [16]. Lately, the resolution of the Schwinger-Dyson formulation of the gap equation has revealed that the effect of the dynamical polarization can significantly lower the critical coupling for exciton condensation, down to a value α c ≈ 0.92 for N = 4 [20].
In this paper we take advantage of the renormalization properties of the Dirac theory in order to assess the effect of different many-body corrections to the excitonic instability. In this respect, the renormalization of the quasiparticle properties can have a significant impact, mainly through the increase of the Fermi velocity at low energies [21,22]. Thus, we will consider the renormalization of the electron-hole vertex accounting for the exciton condensation in the ladder approximation, supplemented by self-energy corrections to electron and hole states. This dressing of the bare quasiparticles will have the result of increasing the critical coupling at which the excitonic instability takes place, going in the same direction as the effect of screening the Coulomb interaction. We will see however that, incorporating the dynamical polarization in the ladder approximation, the screening effects are softened at N = 4, leading to values of the critical coupling below those corresponding to the nominal interaction strength in isolated free-standing graphene.
Our main interest is to study the behavior of the vertex for the operator ρ m (r) = ψ(r)ψ(r). This gives the order parameter for the exciton condensation, and the signal that it gets a nonvanishing expectation value can be obtained from the divergence of T ρ m (q, t)ρ m (−q, 0) . The singular behavior of this susceptibility can be traced back to the divergence of the vertex for ρ m (q)ψ(k + q)ψ † (k) at q → 0. We will denote the vertex in this limit (setting also the corresponding frequency ω q = 0) by Γ(k, ω k ). In the ladder approximation, depicted diagrammatically in Fig. 1, the vertex is bound to satisfy the equation where V (p, ω p ) stands for the Coulomb interaction. We will deal in general with the RPA to screen the potential, so that V (p, ω p ) = e 2 /(2|p| + e 2 χ(p, ω p )), in terms of the polarization χ for N two-component Dirac fermions. Eq. (2) is formally invariant under a dilatation of frequencies and momenta, which shows that the scale of Γ(k, ω k ) is dictated by the high-energy cutoff Λ needed to regularize the integrals. The vertex acquires in general an anomalous dimension γ ψ 2 , which governs the behavior under changes in the cutoff [23] We recall below how to compute γ ψ 2 , showing that it diverges at a critical value of the interaction strength. This translates into a divergence of the own susceptibility at momentum transfer q → 0, providing then the signature of the condensation of ρ m (r) = ψ(r)ψ(r) and the consequent development of the excitonic gap. Self-energy corrections to ladder approximation.-We deal first with the approach in which electrons and holes are dressed by self-energy corrections, while the Coulomb interaction in (2) is screened by means of the static RPA with χ(p, 0) = (N/16)|p|/v F . It is known that graphene remains a conventional Fermi liquid even at the charge neutrality point, with a quasiparticle weight that does not vanish at the Fermi level [24]. The most important self-energy effect comes from the renormalization of the Fermi velocity at low energies [25], and this is the feature that we want to incorporate in our analysis, identifying v F in Eq. (2) with the Fermi velocity dressed by selfenergy corrections.
The electron self-energy corrections, as well as the terms of the ladder series, are given by logarithmically divergent integrals that need to be cut off at a given scale Λ. Alternatively, one can also define the theory at spatial dimension d = 2 − ǫ, what automatically regularizes all the momentum integrals. Eq. (2) then becomes where v F (p) is the Fermi velocity dressed with the selfenergy corrections, κ = 1 + N e 2 /32v F , and e 2 0 is related to e 2 through an auxiliary momentum scale ρ such that e 2 0 = ρ ǫ e 2 .
In the ladder approximation, the Fermi velocity gets a divergent correction only from the "rainbow" selfenergy diagram with exchange of a single screened interaction [25]. The dressed Fermi velocity becomes The expressions (4) and (5) are singular in the limit ǫ → 0. The most convenient way to show that all the divergences can be renormalized away is to resort at this point to a perturbative computation of Γ(k, 0).
The solution of (4) can be obtained in the form with λ 0 = e 2 0 /4πκv F . Each term in the sum can be obtained from the previous one by noticing that At each given perturbative level, the vertex displays then a number of poles in the variable ǫ. The crucial point is that these divergences can be reabsorbed by passing to physical quantities such that v F = Z v (v F ) ren and ψψ = Z ψ 2 (ψψ) ren (the scale of the single Dirac field is not renormalized in the ladder approximation).
The renormalized vertex Γ ren = Z ψ 2 Γ can be actually made finite with a suitable choice of momentumindependent factors Z v and Z ψ 2 . Z v must be chosen to cancel the 1/ǫ pole in (5), and it has therefore the simple On the other hand, we have the general structure The position of the different poles must be chosen to enforce the finiteness of Γ ren = Z ψ 2 Γ in the limit ǫ → 0. The computation of the first orders of the expansion gives for instance the result where the series are written in terms of the renormalized coupling λ ≡ ρ −ǫ Z v λ 0 The physical observable in which we are interested is the anomalous dimension γ ψ 2 . The change in the dimension of Γ ren comes from the dependence of Z ψ 2 on the only dimensionful scale ρ, being γ ψ 2 = (ρ/Z ψ 2 )∂Z ψ 2 /∂ρ [23]. The bare theory at d = 2 does not know about the arbitrary scale ρ, and the independence of λ 0 = ρ ǫ λ/Z v on that variable leads to The anomalous dimension is then [26] γ ψ 2 = ρ ∂λ ∂ρ In the derivation of (11), it has been implicitly assumed that poles in the variable ǫ cannot appear at the righthand-side of the equation. For this to be true, the set of equations c ′ n+1 = c n c ′ 1 − b 1 c ′ n must be satisfied identically [26]. Quite remarkably, we have verified that this is the case, up to the order λ 7 we have computed the coefficients in (8). This is the proof of the renormalizability of the theory, which guarantees that physical quantities like γ ψ 2 remain finite in the limit ǫ → 0.
From the practical point of view, the important result is the evidence that the perturbative expansion of c 1 (λ) approaches a geometric series in the λ variable. It can be checked that the coefficients in the expansion grow exponentially with the order n, in such a way that A lower bound for d can be obtained from the first orders in c 1 (λ). This estimate tends to increase as it is made from higher orders in the expansion. The assumption of scaling with the order n allows us to estimate a radius of convergence λ c ≈ 0.49 (to be compared with the value in the approach neglecting self-energy corrections, which leads to λ c ≈ 0.45 [18], in close agreement with the result of Ref. 16). The critical coupling in the variable λ = α/κ can be used to draw the boundary for exciton condensation in the (N, α) phase diagram, represented in Fig. 2. For N = 4, we get in particular the critical coupling α c ≈ 2.09, significantly above the critical value that would be obtained from the radius of convergence without self-energy corrections (α c ≈ 1.53). Dynamical screening in the ladder approximation.-In the framework of the ladder approximation, one can also study the effect of the dynamical screening of the Coulomb interaction. We can go beyond the static RPA by taking into account the full effect of the frequencydependent polarization, which for Dirac fermions takes the form χ(p, ω p ) = (N/16)p 2 / v 2 F p 2 − ω 2 p [25]. This expression can be introduced in Eq. (2) to look again for self-consistent solutions for the vertex Γ(k, ω k ). While this problem does not afford an analytic approach of the type shown before, one can resort to numerical methods for the resolution of the integral equation. In this procedure, we find again that there is a critical coupling at which Γ(k, ω k ) blows up, marking the boundary between two different regimes where the integral equation has respectively positive and negative (unphysical) solutions. In practice, we have solved the integral equation (2) by defining the vertex in a discrete set of points in frequency and momentum space. One can take as independent variables in Γ(k, ω k ) the modulus of k and positive frequencies ω k , and we have adopted accordingly a grid of dimension l × l covering those variables. The advantage of this approach is that the number l plays the role of cutoff, making straightforward to assess the effect of higher frequencies and momenta as l is increased. We have solved the integral equation in l × l grids with l running up to a value of 200, for which it is still viable to invert a matrix of dimension l 2 . One can rely moreover on the scale invariance of the theory to find the trend at larger values of l, as the critical coupling α c must obey a well-defined finite-size scaling law as a function of the cutoff α c (l) = α c (∞) + c/l ν .
For a given value of N , we have determined the critical point at which the vertex Γ(k, ω k ) blows up. The value of α c (l) for our largest l provides an upper bound for the critical coupling in the continuum limit, as α c (l) turns out to be always a decreasing function of the variable l. On the other hand, the use of the above finite-size scaling law allows to estimate α c (∞) . We have chosen to represent in Fig. 2 the conservative upper bound α c (200) as a function of N . In marked difference with other approaches, we observe that now a critical coupling always exists, no matter how large the value of N may be. For N = 4 corresponding to graphene, we get the values α c (200) ≈ 1.08 and α c (∞) ≈ 0.99.
We see that the value of the critical coupling obtained upon dynamical screening of the interaction is substantially smaller than the value found for N = 4 in the static approximation. This agrees with the results obtained in Ref. [20], where the resolution of the gap equation was accomplished taking into account the frequencydependent polarization. The critical coupling obtained there for N = 4, α c ≈ 0.92, is actually very close to our estimate α c (∞) ≈ 0.99. These values are also close to the critical coupling α c ≈ 1.08 found in lattice Monte Carlo simulations [14], suggesting that the consideration of the dynamical screening provides a most sensible approximation to the excitonic instability.
Conclusion.-We have shown that the various manybody corrections used to dress the electron quasiparticles and the Coulomb interaction can have significant impact on the excitonic instability. The effects of the electron self-energy corrections and the electron-hole polarization have an important role in reducing the strength of the instability. We have seen however that, as anticipated in Ref. [20], the simple static approximation overestimates the screening effects, and that a more accurate approach to the problem requires the consideration of the dynamical screening of the interaction.
It is puzzling that, if we were to use the nominal values of the parameters appropriate for graphene, we would arrive at the conclusion that an isolated free-standing layer of the material (for which α ≈ 2.2) should be in the phase of exciton condensation. This is at odds with the absence of any experimental observation of a gap in suspended graphene samples. Our many-body analysis shows that the only possible relevant effects that have been dismissed are those related to the scaling of the quasiparticle parameters. In this respect, the growth of the Fermi velocity at low energies [24] can have a deep impact to prevent the excitonic instability [22,27]. This effect, expressed by the scaling law (10) at ǫ = 0, has been already observed in experimental samples of graphene at very low doping levels [28]. It is quite plausible that, at the lowenergy scales where the gap could develop (about three orders of magnitude below the scale of the high-energy cutoff), the scaling of the Fermi velocity may have driven the effective coupling to such small values that the excitonic instability cannot then proceed. This can be one more consequence of the nontrivial scaling properties of the theory of Dirac fermions, implying that the electrons in graphene approach a noninteracting regime as they are observed at energies arbitrarily closer to the charge neutrality point. | 2011-04-11T16:40:05.000Z | 2011-03-18T00:00:00.000 | {
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253237465 | pes2o/s2orc | v3-fos-license | The trigonal structure as a reference to access the spontaneous polarization of wurtzite crystals
The spontaneous polarization of wurtzite III-V nitrides XN (X=Al, Ga, In) and II-VI oxides YO (Y=Be, Zn) is investigated via first-principles computational methods. The modern treatment defines this quantity as the polarization difference between the investigated system and an appropriate reference state. We demonstrate that the trigonal structure can be used as a reference to determine the spontaneous polarization of wurtzite materials. We compare the current values with the widely-known zincblende results reported in the literature and find a very good agreement. It is shown that the electronic contribution of polarization is greater than the ionic one. Furthermore, we reproduce the experimental value of the spontaneous polarization of wurtzite GaN reported in a previous study. In order to do so, we calculate the spontaneous polarization for each type of stacking faults using periodic supercells and the Berry-phase method. This theoretical analysis leads to a value nearly identical to the experimental measurement.
I. Introduction
Due to their intrinsic low-symmetry, wurtzite crystals are characterized by the existence of a built-in electric polarization, called spontaneous polarization, which persists even in equilibrium [1]. The spontaneous polarization manifests itself as bound charges and internal electric fields in the layers of heterostructures, significantly affecting the properties and performance of semiconductor devices, such as light-emitting diodes, lasers, and high mobility transistors [2,3]. Experimental measurements of the spontaneous polarization are very limited, simply because such measurements are too challenging [4]. Part of this difficulty is due to the fact that wurtzite crystals are pyroelectrics: the orientation of their polarization is fixed and cannot be reversed [4]. In ferroelectrics, on the other hand, application of a sufficiently intense electric field inverts the polarization vector. This polarization-reversal effect allows an accurate measurement of the spontaneous polarization and is the basis of many important applications [5]. Except for GaN [6], no direct determination for the spontaneous polarization is available, and the experimental values for the majority of wurtzite materials are still unknown. Because of this, theoretical calculations are currently used to provide polarization values in order to interpret the results of experiments and for device modeling.
The theoretical framework in which the polarization of solids is studied is the Berry-phase method, developed in the 90s by Resta, Vanderbilt and King-Smith [7,8]. This method is based on two fundamental insights. First, only polarization differences between two states are accessible and rigorously defined, experimentally as well as theoretically. Second, the difference in polarization is directly related to a quantum geometric phase of the electronic wavefunctions known as the Berry phase [9]. In the specific case of the spontaneous polarization, the two states correspond to a noncentrosymmetric structure (wurtzite in the present case) and its centrosymmetric counterpart, also referred to as the reference structure [10,11]. For materials crystallizing in the wurtzite phase, the cubic zincblende structure has been used in the literature as a reference to calculate the spontaneous polarization [12][13][14][15][16]. In this paper, we propose the trigonal structure as another reference in terms of which the spontaneous polarization of wurtzite crystals is defined. We show that the values that result using this reference are very close to those obtained by zincblende. Furthermore, the trigonal structure can be easily extended to include the effect of stacking faults on the spontaneous polarization. Because of their importance in modern technology, the binary III-V nitrides and II-VI oxides are chosen as case-studies to elucidate the equivalence between the two reference structures in determining the spontaneous polarization of wurtzite compounds.
As mentioned above, the spontaneous polarization of GaN was directly obtained from experiment [6]. This was achieved by employing the energy of excitons bound to the stacking faults in the crystal. Here, we reproduce the reported value using first-principles calculations. Specifically, we compute the spontaneous polarization associated with different types of stacking faults in wurtzite GaN. As will be shown later on, the spontaneous polarization of the perfect (defectless) crystal is much greater than that in the presence of stacking faults. This discrepancy is one of the issues that will be addressed in the present work. The rest of the paper is organized as follows. In section II, we describe both wurtzite and trigonal structures, and provide the numerical details of the simulation. Section III presents the results of the paper: section III.1 deals with the structural optimization needed to perform the calculations. Section III.2 gives the spontaneous polarization values referenced to the trigonal structure. In section III.3 we discuss the electronic and ionic contributions. We present the results of the spontaneous of stacking faults in GaN and explain the procedure carried out to determine them in section III.4. Finally, in section IV a conclusion is drawn which summarizes our main results.
II.1 Wurtzite and trigonal structures
The wurtzite structure has a hexagonal Bravais lattice (space group P63mc or 186) with four atoms per unit cell and two lattice constants: the edge length a of the hexagon base and the height c of the hexagonal prism. However, there is a third structural degree of freedom, a dimensionless internal parameter u that characterizes the cation-anion bond length parallel to the c-axis [14]. A schematic of the wurtzite unit cell is illustrated in figure 1 (a). The cations occupy (1/3,2/3,0) and (2/3,1/3,1/2) positions, while the anions are located at (1/3,2/3,u) and (2/3,1/3,u+1/2). The two types of atoms are tetrahedrally coordinated: each atom is surrounded by four atoms of the other type situated at the corners of a tetrahedron and vice versa. The three bonds in the basal plane are equivalent, but the bond along the c-axis is a little bit longer. This makes the wurtzite structure noncentrosymmetric, i.e. it lacks a center of inversion. This symmetry reduction, in addition to the ionic nature of the bonding, are the physical origin of the spontaneous polarization [17]. In such a way, the atomic positions are identical to zincblende using a different space group: (i) the bonding is tetrahedral with the four bonds having the same length, and (ii) the stacking of atomic planes is in the sequence ABC along the [111] direction, which is in opposition to the wurtzite system, where the stacking is AB along [0001]. Because of these similarities, the trigonal structure is sometimes referred to as the six-atom [111]-oriented zincblende structure [18]. Our major finding is that the trigonal structure is well suited for the derivation of the spontaneous polarization of wurtzite materials, yielding similar results as the ordinary zincblende phase.
II.2 Computational details
The first-principles results presented in this work are obtained using density functional theory (DFT) [19] within the framework of the Kohn-Sham (KS) algorithm [20] as incorporated in the computer code WIEN2k [21]. In all calculations, the exchange-correlation contribution to the total energy is described by the combination of the general gradient approximation [22] and the modified Becke-Johnson approach [23,24] (GGA+mBJ). To solve the KS equations we rely on the full-potential linearized augmented plane-wave (FP-LAPW) method [21,25], which consists of splitting the unit cell using a spherical boundary (called muffin-tin sphere) around the atomic cores. This allows us to expand the solutions on different basis sets inside and outside the spheres: nonlocalized plane waves on the outside, and localized atomic-like orbitals inside. In principle, these expansions run to infinity, but in a real calculation they have to be limited at some point, this is called the expansions cutoff. In our study the development on atomic orbitals is up to a maximum angular momentum lmax=10, and a cutoff of RMT Kmax=8.5 is used for the plane-wave expansion, where RMT is the radius of smallest muffin-tin sphere (see below), and Kmax represents the norm of largest wave vector.
For energy calculations, we need to solve the Schrödinger-like KS equations everywhere in the first Brillouin zone (BZ) of the crystal. In practice, we do it for a finite number of k-values, and get a value for the energy at each k. The tetrahedron method is employed to perform the integration over the BZ by taking a dense mesh of 1000 k-points (a uniform sampling of 10×10×10). These values of the expansion cutoffs and k-points are found to be enough for the convergence of the total energy and electric charge to an accuracy of about 10 −4 Ry and 10 −3 e, respectively.
FP-LAPW is an all-electrons technique, meaning it considers all electrons self consistently in a full-potential treatment [21,25]. The core electrons are fully confined within the muffintin spheres while valence electrons are not. The radii RMT of these atomic spheres for different elements are chosen as follows: 1.5, 1.8, 2, 2.1, 1.6, 1.4, and 2.05 Bohr for N, Al, Ga, In, O, Be and Zn, respectively. We take the borderline to distinguish between core and valence electrons to be −6 Ry, i.e. all orbitals with an energy more negative than this value are core states.
The computation of polarization properties is carried out via the code BerryPI [26], which allows to calculate the electronic and the ionic contributions. The electronic part of the spontaneous polarization is evaluated as a Berry phase of the cell-periodic Bloch functions. For the sake of consistency and maintaining the same degree of convergence, we kept the number of k-points in BerryPI the same as in total-energy calculations.
III.1 Structural optimization
In order to determine the spontaneous polarization for each material, two structures must be carefully prepared. These are the wurtzite and the reference trigonal structures. For the wurtzite system, the determination of structural properties is performed by means of totalenergy minimization process [27]. The equilibrium lattice constants a and c are obtained from a fit of the system energy as a function of unit cell volume and c/a-ratio. The internal parameter u is obtained by the relaxation of atomic positions according to the Hellman-Feynman theorem [28], such that the convergence criterion on the forces is 0.1 mRy/Bohr. The results of the structural optimization are shown in table 1 for each wurtzite compound. We find an overall agreement between our calculated values and those obtained by experiment [29][30][31]. The values of lattice constants a and c obtained by GGA+mBJ tend to be higher than those obtained experimentally. The relative differences are only about 1%, which is typical for wellconverged DFT-calculations. The theoretical internal parameters agree very well with the experimental ones. For all wurtzite crystals, u is slightly greater than the ideal value 0.375. It is not necessary to fully optimize the geometry of the trigonal structure, because its in-plane lattice parameter a is required to be the same as that of the wurtzite phase [16], such that the mismatch Δa/a is identically zero. As for the out-of-plane lattice constant c, it is obtained from the lattice-constant ration c/a as follows. The ideal hexagonal c/a equals √8/3 [32]. Since the trigonal system has three atomic layers [see Fig. 1 (b)], and the thickness of one layer is 0.5c [33], the trigonal ratio c/a is simply 1.5√8/3. With a bit of algebra, the lattice constant c is given by c=a√6. We report in table 1 the values of the trigonal lattice parameters. Note that unlike the wurtzite phase the trigonal one does not contain internal degrees of freedom.
III.2 Spontaneous polarization
Once the structures are prepared, we perform a self-consistent field (SCF) calculation to determine the wavefunctions, from which the spontaneous polarization Psp is evaluated with the Berry-phase technique. This is done, as outlined in section I, by computing the difference between the polarization of the relaxed wurtzite and the trigonal structures: where WZ and T denote the wurtzite and trigonal polarizations, respectively. Within the Berry phase scheme, PWZ and PT are usually called the formal polarizations, and the difference PWZ -PT is the effective polarization [10,11]. We stress that this difference, not the formal polarization by themselves, is the quantity of interest. In table 2 we list the formal polarization as well as the effective spontaneous polarization values for both the nitrides and oxides referenced to the trigonal structure, together with the results of previous calculations that are based on cubic zincblende for the sake of comparison. [15], c Ref. [14], d Ref. [35] It appears that wurtzite nitrides and oxides are highly polarized: their spontaneous polarization is quite large, only one order of magnitude smaller than that of typical ferroelectrics [26]. The spontaneous polarization has a negative sign and is nonzero only along the c-axis, which corresponds to the [0001 ̅ ] direction. This is expected since the intrinsic asymmetry of the bonding is parallel to this specific direction. In the present theory, the polarization is only unambiguously defined if its value is much smaller than the polarization quantum ec/V [11], where c is the lattice constant in the direction of the polar axis and V is the volume of the wurtzite unit cell. The value of this quantum is found to be around 2 C/m² for all compounds, two orders of magnitude greater than the calculated values of the spontaneous polarization, indicating that our results are physically meaningful.
As can be readily seen from the data in table 2, our values derived using the trigonal structure are in good agreement with the values reported in the literature obtained with zincblende. This agreement holds very well for the nitrides. There is a moderate scatter in the theoretically determined spontaneous polarizations for the oxides. However, our values are within the range of those reported in the literature. We have therefore verified that the trigonal and zincblende reference structures provide almost similar values when the spontaneous polarization of wurtzite crystals is concerned. As a final point, we investigate the linearity of the spontaneous polarization as a function of the internal parameter u. For this purpose, the spontaneous polarization is evaluated for internal strains ɛu ranging from 0 to 5%. The internal strain is given by = − 0 0 ⁄ , where 0 is the equilibrium internal parameter and > 0 . The hexagonality of the structure is held constant throughout the calculation, since u can take on different values without altering the symmetry of the unit cell. The results of this investigation are depicted in figure 4. The polarization is generally largest in AlN, smallest in ZnO, and intermediate in BeO, GaN and InN. The spontaneous polarization is observed to vary linearly and remains linear over the entire range of internal strain. The same result was previously obtained in the case of ferroelectric perovskites [36,37], which makes this kind of linearity a general feature of systems exhibiting a spontaneous polarization.
III.3 Electronic and ionic contributions
Electric polarization can be decomposed into two distinct terms as follows [10,11]: where Pele and Pion are respectively the electronic and ionic contributions to the spontaneous polarization. The Berry-Phase method is only used to evaluate the electronic part of polarization, and has nothing to do with the determination of the ionic part. This is because electrons are described by quantum wavefunctions, while ionic cores are treated as classical point charges. To provide further insight into understanding the origin of the strong polarization in wurtzite crystals, we compute each term separately. The calculated contributions using the trigonal phase as a reference are reported in table 3. We observe that the two terms are of opposite sign. Specifically, the electronic part is negative while the ionic one is positive. This sign alternation seems typical for all tetrahedrally bonded materials. In addition, they have different magnitudes, with the electronic term being the largest, resulting in a negative value of the total polarization. One can safely anticipate, as remarked by Ahmed et al., that in nonpolar crystals the absolute value of each of the terms is roughly the same, so that they tend to cancel each other, yielding zero net polarization [26].
In contrast, this cancellation is much less effective in wurtzite compounds, which is the reason of their large polarization.
A. Stacking faults in wurtzite crystals
The wurtzite structure in which GaN crystallizes is formed by the stacking of atoms in the sequence ..AB.. along the [0001] direction, where every layer is surrounded by two identical layers (e.g. ABA). In this case A-B bonds are called hexagonal bonds [38,39]. The zincblende (or trigonal) structure, on the other hand, can be described by the stacking sequence ..ABC.. along the [111] direction, in which the two layers surrounding a given layer are different (e.g. ABC), and the A-B bonds are called cubic bonds. Stacking faults can then be viewed as local deviations of the perfect wurtzite stacking sequence. In other words, stacking faults are embedded cubic units surrounded by a hexagonal matrix. There are three types of basal-plane stacking faults reported in the literature: two intrinsic stacking faults of type-I and -II, and one extrinsic stacking fault. The type-I stacking fault originates from one violation of the wurtzite stacking rule, it introduces one cubic bond. The type-II stacking fault results from two violations of the normal stacking role and introduced two cubic bonds. Finally, the extrinsic stacking fault contains an additional C layer inserted in the midst of the original ..AB.. stacking sequence. This stacking fault introduces three cubic bonds [38,39].
In this work we reproduce the reported experimental value of the spontaneous polarization of GaN via ab-initio calculations, to be described shortly. However, we first present a brief overview of the experimental method used in Ref. 6. This method is based on a model in which the stacking faults are treated as a plate capacitor. The output of the measurement is the magnitude of the sheet charge that built-up at the wurtzite/zincblende interface, which is directly related to the spontaneous polarization of the low-symmetry structure: In the above expression, Δd is the difference in thickness between each of the stacking faults, ε is the dielectric constant and ε0 is the permittivity of free space. The potential change ΔV in this case is equal to the difference in transition energies between the stacking faults. The latter is the key ingredient of this model: the use of equation 1 for determining Psp requires at least a rough estimate of the transition energies of all types of stacking faults. This was done by two sets of spectroscopic measurements performed on samples of strain-free GaN microcrystal. Interested readers are referred to Refs. [6,33] for more details.
B. Supercell structure
In order to model the stacking faults, we use periodic supercells containing 20 atoms, i.e. 10 atomic layers, in a construction of 1×1×5 wurtzite unit cells. The different types of stacking faults are achieved by a proper manipulation of specific atomic positions applied to the original wurtzite stacking sequence as described above. The adopted supercells of the stacking faults in GaN are presented in figure 3. Due to imposing periodic boundary conditions, the beginning and the end of the supercells along the c-axis have to match. This has the effect of doubling the number of cubic bonds for each type of stacking faults. Indeed, the type-I stacking fault supercell shown in Fig. 3(b) contains two cubic bonds instead of one. The same remark can be made for type-II and extrinsic stacking fault supercells, in which there are four and six cubic bonds respectively (instead of two and three). For supercell calculations, which are more computationally expensive, the wave-plane expansion cutoff and the sampling in kspace reported in Sec. II.2 are reduced to 7 and 300, respectively.
C. Results
We begin by determining the equilibrium structure of each stacking fault. We find that the presence of these defects has a negligible effect on the lattice parameters of wurtzite GaN reported in table 1, which means that the cubic units are almost perfectly lattice-matched to the hexagonal host. This result is in accordance with a previous study of stacking faults in nitrides [38]. Also, the atomic positions in the neighborhood of the stacking faults have been relaxed. Using the theoretical approach followed throughout this work, the spontaneous polarization of each type of stacking faults is calculated as the difference: where is the formal polarization of the wurtzite supercell containing the stacking fault, and PT is the trigonal formal polarization. The calculated values for the intrinsic and extrinsic stacking faults are given in the histogram of figure 4. Also included is the Psp value of the ideal structure, where "ideal" refers to the supercell without stacking faults [Fig3. (a)]. It is important to observe the trend followed by the stacking faults: the spontaneous polarization decreases in magnitude with the number of cubic bonds, i.e. from type I to type II and, more significantly, to extrinsic stacking fault. Indeed, the extrinsic stacking fault has the closest structure to the nonpolar zincblende phase (with three cubic bonds); consequently, its polarization is the smallest among the stacking faults studied here. The supercell value of the perfect crystal is the same as the one obtained using an ordinary unit cell. This structure does not contain any cubic bonds by construction, this is why it has the maximum value of the spontaneous polarization. To confirm the validity of the plate capacitor model, equation 3 was used in [6] to estimate the spontaneous polarization for the extrinsic stacking fault. In this case, ΔV and Δd are obtained from self-consistent Poisson-Schrödinger calculations in Ref. [6]. This results in a value of -0.018 C/m², which is very close to our value of -0.016 C/m² for the same stacking fault (see Fig. 4).
The spectroscopically observed ΔV in Eq. 3 is actually the average value of the transition energy difference between the three types of stacking faults. So in a sense, the recommended experimental value of -0.022 C/m² is the spontaneous polarization of type I, type II and extrinsic stacking faults combined. Motivated by this, we compute the average of the three spontaneous polarization values given in figure 4. The value that we get is almost identical to the experimental one, namely -0.021 C/m². This discussion shows that assigning the experimental value of [6] or the average theoretical value reported here to GaN is somehow misleading: -0.022 C/m² or -0.021 C/m² is not the spontaneous polarization of the perfect defectless GaN, it is rather the value associated with the different types of stacking fault of this material.
IV. Conclusion
In this paper, we have performed a theoretical study of the spontaneous polarization in wurtzite crystals based on density functional theory in combination with the Berry-phase technique. We have chosen to investigate this property for the III-V nitrides and II-VI oxides. These are technologically significant materials and a fundamental understanding of their polarization effects is of central importance. Our results demonstrate that the trigonal structure allows a consistent determination of the spontaneous polarization of wurtzite compounds: the values obtained using this structure as a reference show an excellent agreement with the theoretical zincblende-based results reported in the literature. We have shown that the electronic and ionic part of the spontaneous polarization are of opposite sign, with the electronic term has the largest effect, indicating that the strong polarization in these materials has its origin from the partial cancellation of the two terms. Also, the spontaneous polarization was found to follow a monotonous linear behavior with the wurtzite internal parameter. Finally, we have reproduced the experimental value of the spontaneous polarization of wurtzite GaN. This was achieved by taking advantage of the stacking faults within the material. Specifically, averaging the spontaneous polarization values obtained for different types of stacking faults gives a result almost identical to the only reported experimental figure. Our theoretical approach is consistent with the experimental procedure, and can be used to determine the spontaneous polarization of other wurtzite crystals as well. We are not aware of any works where the spontaneous polarization of wurtzite stacking faults is reported. Indeed, the lack of such information was an important factor in motivating the present first-principles study. | 2022-11-01T01:16:13.518Z | 2022-10-31T00:00:00.000 | {
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231733417 | pes2o/s2orc | v3-fos-license | Molecular Mechanisms of L1 and NCAM Adhesion Molecules in Synaptic Pruning, Plasticity, and Stabilization
Mammalian brain circuits are wired by dynamic formation and remodeling during development to produce a balance of excitatory and inhibitory synapses. Synaptic regulation is mediated by a complex network of proteins including immunoglobulin (Ig)- class cell adhesion molecules (CAMs), structural and signal-transducing components at the pre- and post-synaptic membranes, and the extracellular protein matrix. This review explores the current understanding of developmental synapse regulation mediated by L1 and NCAM family CAMs. Excitatory and inhibitory synapses undergo formation and remodeling through neuronal CAMs and receptor-ligand interactions. These responses result in pruning inactive dendritic spines and perisomatic contacts, or synaptic strengthening during critical periods of plasticity. Ankyrins engage neural adhesion molecules of the L1 family (L1-CAMs) to promote synaptic stability. Chondroitin sulfates, hyaluronic acid, tenascin-R, and linker proteins comprising the perineuronal net interact with L1-CAMs and NCAM, stabilizing synaptic contacts and limiting plasticity as critical periods close. Understanding neuronal adhesion signaling and synaptic targeting provides insight into normal development as well as synaptic connectivity disorders including autism, schizophrenia, and intellectual disability.
INTRODUCTION
Formation of synaptic contacts, pruning of axonal and dendritic processes, and elimination of synapses occurs during development of the mammalian brain and is vital for establishment of neuronal circuitry. These dynamic responses occur within both excitatory and inhibitory neurons in early postnatal life, are often associated with critical periods of plasticity, and can be dependent on neural activity. It is thought that overproduction of neuronal processes and nascent synapses followed by selective pruning, serves to stabilize active connections and eliminate less active ones, resulting in an appropriate excitatory-inhibitory balance in cortical networks. Identification of postnatal mechanisms for regulating synapse density is important for understanding how normal circuits are formed, as well as providing insight into how these circuits may be altered in neurodevelopmental disorders such as autism, schizophrenia, bipolar disorder and intellectual disability (Forrest et al., 2018).
Defective pruning of dendritic spines and excitatory synapses in the human brain is a leading hypothesis to explain increased spine density in frontal cortical areas, and social and cognitive impairments seen in autism and Fragile X syndrome (Hutsler and Zhang, 2010;Tang et al., 2014). Excessive spine pruning in the prefrontal cortex may conversely contribute to decreased spine density in schizophrenia and bipolar disorder (Konopaske et al., 2014;Phillips and Pozzo-Miller, 2015). Recent evidence has revealed that inhibitory cortical connections display significant levels of remodeling during the juvenile to adult transition. Among the numerous types of interneurons, parvalbumin (PV)expressing basket cells display active remodeling in postnatal development at the perisomatic region of cortical pyramidal cells (Sullivan et al., 2020). These interneurons regulate synchronous firing, gamma rhythms, and working memory, all of which are impaired in schizophrenia (Gonzalez-Burgos et al., 2015).
Immunoglobulin (Ig) class cell adhesion molecules are transmembrane glycoproteins that are widely expressed in the mammalian nervous stem where they regulate diverse aspects of brain development. L1-CAMs, NCAM, as well as Synaptic Cell Adhesion Molecules (SynCAM1-4) are among the most wellstudied regulators of synaptic development and plasticity. This review focuses on the role of this group of adhesion molecules in synapse remodeling and stabilization of excitatory and inhibitory connections during maturation of neural circuits. Other recent reviews cover the role of IgCAMs in synapse formation (Sytnyk et al., 2017;Cameron and McAllister, 2018).
Single null mutant mice lacking NrCAM, CHL1, Npn2, PlexA3, or Sema3F display elevated spine and excitatory synapse density as shown in several neocortical areas Demyanenko et al., 2014;Mohan et al., 2019b). This phenotype is restricted to apical dendrites, where Npn2 is preferentially localized (Tran et al., 2009). Selective spine pruning on apical dendrites is of interest with regard to cortical network establishment, as apical dendrites differ from basal dendrites in responding more robustly to intra-cortical and thalamocortical inputs, and having distinctive temporal plasticity rules (Gordon et al., 2006;Shigematsu et al., 2016). Excitatory neurotransmission is increased in NrCAM and CHL1-deficient prefrontal slices, consistent with cortical hyperexcitability (Demyanenko et al., 2014;Mohan et al., 2019b). It is of interest that Sema3B (Mohan et al., 2019b) and Sema3F (Orr et al., 2017;Wang et al., 2017) are secreted in an activity-dependent manner, as shown in cultures, raising the possibility that limited diffusion of Sema3 ligands from spines may prune less active, immature neighbors to refine cortical circuits. Indeed, spines with immature, thin morphology are selectively pruned by Sema3F and Sema3B (Demyanenko et al., 2014;Mohan et al., 2019b). The Sema3F/Npn2/PlexA3 complex also plays an adult role in cortical neurons, facilitating homeostatic scaling of synaptic strength by interacting with the glutamate receptor GluA1 (Wang et al., 2017). Homophilic binding in cis and trans is mediated principally by the Ig2 domain, which is present within a folded horseshoe conformation of Ig1-4. The L1 Ig1 domain binds heterophilically to the Sema3A co-receptor Neuropilin-1 (Npn1). Npn1/2 consist of 2 CUB domains (a1, a2), 2 coagulation factor V/VII domains (b1, b2) and a meprin-A5-mu domain (MAM). Other L1-CAMs Close Homolog of L1 (CHL1) and NrCAM have similar structures and carry out related functions. The NrCAM Ig1 domain constitutively binds Npn2 at its a1 domain and mediates responses to Sema3F in complex with PlexA3. Activation of the intrinsic Rap-GAP activity of PlexAs downregulates Rap1-GTPase. CHL1 binds Npn2 and mediates similar responses to Sema3B in complex with PlexA4. All L1-CAMs reversibly bind AnkyrinB/G (AnkB/G), a spectrin-actin adaptor protein, at a conserved motif FIGQ/AY in the cytoplasmic domain.
The molecular mechanism leading to spine loss is best understood for the Sema3F holoreceptor. Binding of NrCAM to Npn2 occurs in cis within the dendritic membrane and is mediated by charged residues within two motifs (TARNER and DDK) in the NrCAM Ig1 domain, which interact with specific residues in the Npn2 a1 domain (Mohan et al., 2019a). Sema3F dimers induce membrane clustering of Npn2 and constitutively associated PlexA3, stimulating the intrinsic Rap-GTPase activating protein (GAP) activity of PlexA3 (Pascoe et al., 2015). This in turn downregulates Rap1 preventing activation of Rap-dependent Rap1-GTP-interacting adaptor molecule (RIAM) and Talin from inducing inside-out signaling of β1 integrins (Mohan et al., 2019a).
Since activation of β1 integrins is known to promote spine stabilization (DePoy et al., 2019), integrin inactivation and loss of adhesion to the extracellular matrix likely contributes to spine pruning. Actin cytoskeletal remodeling resulting from these effector pathways serves to collapse the affected spines.
NEUROPLASTICITY OF INHIBITORY SYNAPTIC CONNECTIONS
The Ig-class adhesion molecule Neural Cell adhesion Molecule (NCAM1) has been extensively studied for its pre-and postsynaptic roles in regulating synaptic development, long term potentiation (LTP), learning, and memory in excitatory pyramidal neurons of the hippocampus and cerebral cortex [reviewed in Sytnyk et al. (2017)]. In pyramidal neurons, NCAM induces synaptogenesis (Dityatev et al., 2004) as well as endocytosis of pre-synaptic vesicles (Shetty et al., 2013). Current focus has shifted to the role of NCAM in regulating connectivity of GABAergic interneurons, in particular the targeting of the basket interneuron inputs to the perisomatic region of cortical pyramidal neurons.
GABAergic interneurons function to limit the activity of pyramidal neurons thus regulating firing, timing, and synchrony of cortical circuits (Hu et al., 2014). Interneurons are highly diverse and express different markers: calcium binding proteins [parvalbumin (PV), calbindin, and calretinin] and neuropeptides [cholecystokinin (CCK), somatostatin, and vasoactive intestinal peptide] (Wamsley and Fishell, 2017). Basket cells are PVexpressing interneurons that synaptically target the soma and proximal apical dendrite of pyramidal cells. Perisomatic inhibitory synapses derive chiefly from PV+ basket cells but also from some CCK+ interneurons (Karson et al., 2009;Wyeth et al., 2010;Hansen et al., 2017;Veres et al., 2017;Horn and Nicoll, 2018). Perisomatic inhibition is vital for pyramidal cell synchrony, working memory, decision making, and social behavior (Lagler et al., 2016;Ferguson and Gao, 2018), all of which are impaired in neuropsychiatric diseases (Dienel and Lewis, 2019). PV-expressing chandelier interneurons target the axon initial segment (AIS) of pyramidal neurons, where action potentials are initiated (Somogyi, 1977). The density of inhibitory boutons derived from chandelier interneurons onto the AIS has been reported to be increased in layer 2/3 of the dorsolateral prefrontal cortex in schizophrenia (Rocco et al., 2017).
A recently identified function for NCAM is in reducing the potential for inhibitory neurotransmission by pruning excess perisomatic inputs from basket interneurons during postnatal development of the prefrontal cortex (Figure 2A). This remodeling occurs in response to repellent EphrinA ligands (EphrinA2, A3, and A5), which bind the EphA3 receptor kinase in complex with the 140 kDa isoform of NCAM (Pillai-Nair et al., 2005;Brennaman et al., 2013;Sullivan et al., 2016Sullivan et al., , 2018Sullivan et al., , 2020. Loss of NCAM, EphA3, or EphrinA2/3/5 in mice increases the density of perisomatic inhibitory boutons on pyramidal neurons in layer 2/3 of the prefrontal cortex (Brennaman et al., 2013). EphrinA5, a glycophosphatidyl inositol (GPI)-linked protein, stimulates co-clustering of NCAM and EphA3 on basket cell terminals, which triggers retraction of the axon terminal by activating RhoA and its principal downstream effector Rhoassociated protein kinase (ROCK1/2) (Brennaman et al., 2013;Sullivan et al., 2016). An integral component of the mechanism, ADAM10 (a disintegrin and metalloprotease-10) cleaves surfacebound NCAM and EphrinA5 to promote detachment and enable EphrinA-induced retraction (Figure 2A, dotted lines) . Laser scanning photostimulation (LSPS) of GABAergic interneurons expressing light-activated channel-rhodopsin in NCAM-deficient brain slices was shown to increase the inhibitory field of these neurons in the prefrontal cortex, consistent with an increase in the number of functional perisomatic synapses (Zhang et al., 2017). Conditional deletion of NCAM in PV-expressing interneurons in mice was recently demonstrated to constrain the extent of postnatal remodeling of perisomatic inhibitory synapses in the prefrontal cortex of homozygous PV-Cre:NCAM flox/flox :tdTomato mice (Sullivan et al., 2020). This study used live imaging in brain slices to FIGURE 2 | Model of NCAM Mediated Remodeling of Inhibitory Synapses. (A) NCAM1 (there is also a lesser studied NCAM2) contains 5 Ig and 2 FNIII domains, and alternative splicing produces 3 major isoforms with different sizes of the cytoplasmic domain. NCAM140 has the smallest cytoplasmic tail, while NCAM180 kDa has an insert in this sequence. NCAM 120 (not shown) is linked to the plasma membrane by a glycophosphatidyl inositol (GPI) tag and is expressed in glia. NCAM Ig1 and Ig2 domains mediate homophilic adhesion in both cis and trans. The extracellular region of all NCAM isoforms can be modified by polysialylation (PSA) on Ig5, which reduces homophilic binding affinity and promotes neural plasticity. NCAM engages the EphrinA receptor tyrosine kinase EphA3 through interaction of NCAM Ig2 with the cysteine-rich domain (CRD) of EphA3. GPI-linked EphrinA5 on pyramidal cell soma binds the ligand binding domain (LBD) of EphA3 and promotes clustering of PSA-NCAM and EphA3 on axon terminals of parvalbumin-expressing basket interneurons. PSA-NCAM, EphA3, and ADAM10 metalloprotease coordinate to regulate EphrinA/EphA-induced retraction of axon terminals of interneurons from the perisomatic region of cortical pyramidal neurons by downstream signaling through RhoA and Rho kinase (Rock1/2). ADAM10 cleaves PSA-NCAM and EphrinA5 (dotted line) to facilitate retraction. (B) SynCAMs1-4 are synaptic adhesion molecules with 3 Ig domains, an extracellular juxta-membrane region, and a conserved cytoplasmic tail that binds. FERM-and PDZ-containing proteins. SynCAMs engage in heterophilic interactions within the family, as well as cis and trans homophilic adhesion. SynCAM1 promotes synaptogenesis of excitatory synapses as well as synaptic maintenance.
Frontiers in Cell and Developmental Biology | www.frontiersin.org reveal that PV+ perisomatic boutons are highly dynamic in wild type prefrontal cortex but much less dynamic in NCAMdeficient cortex. The PV-Cre: NCAM conditional mutant mice also exhibited impaired inhibitory neurotransmission, working memory, and social behavior (Sullivan et al., 2020).
Polysialylation (PSA) of NCAM on the Ig5 domain is a developmental modification that has a prominent influence on plasticity in cellular responses, including neuronal migration (Schuster et al., 2020) and modulation of extrasynaptic NMDA receptors (Sytnyk et al., 2017;Varbanov and Dityatev, 2017). Polysialylation of NCAM is achieved through the expression and activation of two poly-sialyltransferases, St8sia II, and St8sia IV (Hildebrandt et al., 2010). The 140 and 120 kDa NCAM isoforms promote the maturation of GABAergic basket cell synaptic fields in the visual cortex (Chattopadhyaya et al., 2013). PSA-modification of NCAM prevents the premature closure of the critical period of visual plasticity by restricting innervation of pyramidal cell soma by basket cell terminals, while non-PSA modified NCAM promotes synaptic targeting of these inputs (Di Cristo et al., 2007). In accord with the dynamic nature of basket cell synaptic puncta in the prefrontal cortex, inhibitory synapses in the visual cortex undergo pronounced GABA-dependent remodeling Wu et al., 2012). Evidence suggests that a GABA activity-dependent "punishing signal" may be produced to eliminate inactive basket cell synaptic contacts (Baho and Di Cristo, 2012;Wu et al., 2012). It is interesting to speculate that EphrinA5 might serve as such a signal, as it is upregulated on the neuronal surface in an activitydependent manner in neuronal cultures (Sullivan et al., 2020). In addition, PSA-NCAM is regulated by activity in vivo (Di Cristo et al., 2007). A plausible scenario is that active inhibitory synapses may prune weaker neighbors through EphrinA/NCAM/EphA3 repellent signaling to fine tune microcircuits during postnatal maturation (Figure 2A). Similarly, local competition has been shown to be involved in homeostatic regulation of excitatory synapses in both developing and mature brain (Turrigiano, 2012;Bian et al., 2015;Kehayas and Holtmaat, 2015;Oh et al., 2015;Stein and Zito, 2019).
There is accumulating evidence for developmental refinement of inhibitory connections in other brain regions. The density of GABA-positive boutons decreases from late adolescence to young adulthood in the auditory cortex (Moyer et al., 2015). Inhibitory terminals from the medial nucleus of the trapezoid body onto the lateral superior olivary nucleus are also eliminated upon maturation (Kotak and Sanes, 2014). Intrinsic interneurons in the thalamic dorsal lateral geniculate nucleus were found to remodel their arbors in response to retinal activity (Charalambakis et al., 2019). In the primary somatosensory cortex (S1), GABAergic inputs from the thalamus transiently integrate into cortical circuits and are refined postnatally, potentially serving to suppress excitability from emerging pyramidal cell connections (Marques-Smith et al., 2016). Inhibitory inputs from local interneurons onto pyramidal cells in S1 also undergo postnatal refinement (Cocas et al., 2016). These effects might be region specific, for example the connection probability of PV+ interneurons in S1 layer 2/3 shows only an insignificant decrease with developmental age (Packer and Yuste, 2011).
SynCAMs (1-4) represent a distinct class of Ig-family adhesion molecules that contribute to synapse formation and stability ( Figure 2B). SynCAM1 participates in formation and stabilization of new synapses through complex homophilic and heterophilic binding interactions (Frei and Stoeckli, 2017). SynCAM1 and SynCAM2 form a trans-synaptic complex that promotes formation and maintenance of excitatory synapses (Fogel et al., 2007). These SynCAM interactions are mediated by site-specific N-glycosylation on the extracellular binding interface. Specifically, the N-glycans in SynCAM2 Ig1 decrease adhesion, while glycosylation in SynCAM2 Ig1 increases adhesion . The cytoplasmic domain of SynCAM1 contributes to synapse stabilization by recruiting FERM-and PDZ-containing scaffold molecules (Figure 2B; Frei and Stoeckli, 2017). SynCAM1 also facilitates spine maturation and stability shown by increased spine density and size in SynCAM1 overexpressing mice (Robbins et al., 2010), although contrasting findings were noted in SynCAM1 null mice . Dentate gyrus neurons from SynCAM1 overexpressing mice also exhibit increased spine density and size, as well as increased excitatory transmission manifested by elevated mEPSC frequency and amplitude (Doengi et al., 2016). Furthermore, SynCAM1 null mice display delayed maturation of the visual cortex, the presence of immature cortical PV+ interneurons, and decreased thalamacortical inputs onto PV+ interneurons (Ribic et al., 2019). Recent studies suggest that SynCAM1 may interact with other Ig-class adhesion molecules such as L1-CAMs and NCAM to mediate synaptic stability, as indicated by proteome mapping of the synaptic cleft in cultured cortical neurons (Cijsouw et al., 2018).
ROLE OF ANKYRIN INTERACTION WITH NEURAL ADHESION MOLECULES IN SYNAPTIC STABILIZATION
Synaptic stabilization is a postnatal mechanism to regulate synapse density by strengthening and maintaining synapses. Though persistence and elimination of spines have been extensively studied, recent research has focused on mechanisms of synaptic structural stability (Meyer et al., 2014). Synaptic stabilization involves the clustering and complexing of synaptic adhesion molecules, which enhances the physical interaction of pre-and post-synaptic membranes. Additional interactions between the L1 family members NrCAM and CHL1 stabilize synapses on GABAergic inhibitory neurons by complexing with sensory derived proteins NB2 (Contactin5) of the Ig superfamily and contactin-associated protein Caspr4 (Ashrafi et al., 2014). CHL1 expression on stellate cells in the cerebellum may also stabilize synapses through heterophilic interactions between Purkinje dendrites or homophilic interactions with Bergman glia in postnatal mice (Ango et al., 2008).
One mechanism by which stability of newly formed synapses is achieved is through the recruitment of scaffolding proteins such as the actin cytoskeletal adaptors Ankyrin B/G and their binding partner β-spectrin to the cytoplasmic domains of L1-CAMS (Figure 3; Jenkins and Bennett, 2001;Doengi et al., 2016; FIGURE 3 | Ankyrin Structure and Subcellular Localization with L1 Interactions. (A) Human Ankyrin B and Ankyrin G isoforms. Ankyrin is comprised of an N-terminal membrane binding domain consisting of 24 Ank repeats that recruit L1-CAMs and ion channels, a spectrin binding domain containing ZU5-N, ZU5-C and UPA subdomains, a death domain (DD), and a C-terminal regulatory tail. Ankyrin B has two major splice variants, isoforms 220 kDa and giant AnkB 440 kDa, which has an insert encoded by neuronal exon 37 (bright green). Ankyrin G splice variants include isoforms of 190, 270, and 480 kDa, the latter two of which have inserts encoded by neuronal partial exon 37 and giant exon 37, respectively. AnkG 270 and 480 also contain a serine-rich domain (yellow), which mediates targeting to the axon initial segment (AIS). (B) AnkG 480 kDa. At the AIS, giant AnkG 480 kDa clusters and tethers proteins, including voltage gated sodium channels (VGSCs), KCNQ2/3 potassium channels, NrCAM, NF186 and L1, to the actin-spectrin cytoskeleton. The AnkG spectrin binding domain binds βIV-spectrin, which links to actin at periodic ring structures. AnkG 480 is also localized to the somato-dendritic compartment of pyramidal neurons. (C) AnkG 190 kDa. AnkG 190 is present in dendritic spines, where it stabilizes AMPA receptors by promoting the formation of complexes of AnkG, AMPA receptors and βIV-spectrin at synapses. (D) AnkB 220 kDa. AnkB is reversibly recruited to the cytoplasmic domain of L1 on pyramidal cell soma at PV+ GABAergic interneuron synapses. This binding is fostered by dephosphorylation of L1 at Tyr1229, whereas phosphorylation of this residue results in loss of Ankyrin binding. AnkB220 is also localized to pyramidal cell dendrites and soma. (E) AnkB 440 kDa. Giant AnkB (440 kDa) binds to both L1 and microtubules in axons. AnkB 440 kDa bundles microtubules and tethers them to the actin-βII spectrin cytoskeleton, which helps maintain structural stability of the axon. Yang et al., 2019). Reversible binding of the L1-CAM cytoplasmic domain to Ankyrin is regulated by dephosphorylation of a tyrosine residue (Y1229) in a conserved motif FIGQ/AY ( Figure 3D; Garver et al., 1997;Yamasaki et al., 1997;Needham et al., 2001;Guan and Maness, 2010). Mutation of Tyr1229 in L1 to histidine occurs in the L1 syndrome of intellectual disability (Hortsch et al., 2014). A mouse model of this disorder harbors an L1 knock-in point mutation (Tyr1229His) that impairs L1-Ankyrin binding (Buhusi et al., 2008). These "L1YH" mice exhibit decreased synapse density of GABAergic interneurons targeting pyramidal cell soma in the prefrontal cortex (Guan and Maness, 2010). L1YH mice also lose the L1-AnkG-β4-spectrin interaction at the AIS (Tai et al., 2019), and L1 null mice exhibit reduced numbers of hippocampal perisomatic inhibitory synapses (Saghatelyan et al., 2004). L1 mutant mice that harbor mutations in nuclear receptor binding motifs show decreased inhibitory GAD67+ and excitatory vGlut+ synaptic puncta on cerebellar Purkinje cells (Kraus et al., 2018). Ankyrin links other membrane and scaffold proteins to the cytoskeleton to further achieve synaptic stability.
Multiple alternative splice variants of Ankyrin B (AnkB) are encoded by the ANK2 gene ( Figure 3A). The AnkB 220 kDa isoform is ubiquitously expressed, and in pyramidal neurons appears to localize to dendritic and soma subcellular compartments (Figure 3D; Kunimoto, 1995). Giant AnkB (440 kDa) has a large neuronal-specific insert encoded by exon 37 and associates with the axonal plasma membrane through L1 engagement ( Figure 3E; Yang et al., 2019). Giant AnkB binds and bundles microtubules (MTs) via a 12-residue motif unique to this isoform, tethering MTs to the actin cytoskeleton ( Figure 3E). This interaction may maintain the structural stability and fasciculation of MTs in axons (Chen et al., 2020). Giant AnkB mutant mice and L1YH mice show transiently increased axon branching, which is likely mediated through MT growth (Yang et al., 2019). Additionally, neuromuscular junctions in Drosophila mutants lacking the giant AnkB isoform (Ank2-L) exhibit increased synaptic retractions and fragmented presynaptic membranes (Weber et al., 2019).
Ankyrin G, encoded by the ANK3 gene, mediates recruitment, coordination, and clustering of multiple proteins at the AIS: voltage gated sodium channels (VGSCs), KCNQ 2/3 potassium channels, Neurofascin 186 (NF 186), and β4spectrin (Figures 3A,B; Jenkins et al., 2015). The AIS, located between neuronal somatodendritic and axon domains, is responsible for the initiation of action potentials by maintaining ion channels and regulation of cargo transport and neuronal polarity (Kole et al., 2008;Rasband, 2010;Leterrier, 2018). Securing these hetero-complexes to the actin/spectrin cytoskeleton allows for proper action potential initiation, excitability regulation and maintenance of distinct axon and somatodendritic compartments (Gulledge and Bravo, 2016;Lazarov et al., 2018;Yang et al., 2019;Goethals and Brette, 2020). The giant 480 kDa AnkG isoform is needed for development and function of the AIS. Knockdown of 480 kDa AnkG in cultured hippocampal neurons diminishes clustering of β4spectrin, VGSCs and NF186 . In exon 37 null mice with a specific deletion of 480kD AnkG, recruitment of AnkG to the AIS and clustering of β4-spectrin, NF186, VGSCs, and KCNQ2 were lost in Purkinje neurons . AnkG 480kD also mediates the stability of GABAergic synapses via interaction with GABA A receptor-associated protein, which inhibits endocytosis in hippocampal neurons . AnkG 480 deletion results in GABAergic synapse loss in the hippocampal CA1 region and the cerebral cortex postnatally . In cortical neuron cultures, it was found that AnkG helps to stabilize AMPA receptors at synapses by promoting the formation of multiprotein complexes consisting of AnkG, AMPA receptors, and β-spectrin ( Figure 3C; Smith et al., 2014). Additional studies on human AnkG in psychiatric disorders have been done (Luoni et al., 2016) and AnkG has been recently reviewed in Salzer (2019).
The association of L1-CAM Neurofascin 186 (NF186) with AnkG is required for formation and stabilization of pinceau synapses in the cerebellar Purkinje neurons (Ango et al., 2004). A NF186 gradient directs basket interneuron axons to the AIS of Purkinje cells during pinceau synapse formation. In Purkinje neurons lacking AnkG, NF186 is uniformly distributed, and basket cell axons lack directional growth toward the AIS during postnatal maturation (Ango et al., 2004). The AnkG 190 kDa isoform is localized to nanodomains where it contributes to spine morphology (Smith et al., 2014). β-spectrin is needed to target AnkG to dendritic spines in cortical neurons of adult mice. Mutation of the AnkG 190 kDa isoform in cortical neurons prevents binding of β-spectrin and targeting of AnkG to spines. AnkG targeting of spines is critical for AMPA receptor clustering and regulation (Smith et al., 2014).
PERINEURONAL NET PROTEINS STABILIZE SYNAPSES, BIND NEURAL ADHESION MOLECULES, AND REGULATE SYNAPTIC PLASTICITY
Extracellular proteins that contribute to synapse stabilization constitute the perineuronal net (PNN) (Figure 4). PNNs contain multiple molecules including chondroitin sulfate proteoglycans (CSPGs) with glycosaminoglycan (GAG) side chains, hyaluronic acid, tenascin-R, and linker proteins, which combine to form a mesh-like structure that surrounds maturing neurons, particularly at the soma of PV+ basket cells (Friedlander et al., 1994;Koppe et al., 1997). The PNN develops postnatally at approximately the same time that synapses are formed and stabilized. Neuronal plasticity decreases as the PNN arises and binds to neuronal surface proteins, stabilizing synaptic contacts (Pizzorusso et al., 2002;Wang and Fawcett, 2012;Carulli et al., 2016). Interestingly, PV+ interneurons that are surrounded by PNNs are more mature than interneurons without PNNs (Carceller et al., 2020), potentially providing greater stability to established perisomatic synapses. Additionally, examining cells following enzymatic digestion of PNNs by Proteus vulgaris chondroitinase ABC (chABC) revealed decreases in the number and stability of perisomatic synaptic puncta in mice ( Figure 4A). chABC treatment of tenascin-R null mice also showed reduced perisomatic inhibition (Bukalo et al., 2001). Similar results were seen in normal rats (Lensjo et al., 2017).
Neurocan is a prominent organizer of the PNN and one of the first CSPGs to be expressed in the maturing neocortex. Early research on neurocan showed its prevalence in the brain (>20% of the soluble CSPGs) and its ability to limit neurite outgrowth in culture (Friedlander et al., 1994). More recent studies have shown that neurocan can effectively block synaptogenesis (Celio and Blumcke, 1994;Celio et al., 1998;Dityatev and Schachner, 2003;McRae et al., 2010;Soleman et al., 2013;Suttkus et al., 2016;Sullivan et al., 2018). During postnatal development, neurocan assembles around processes, dendritic spines, and soma of pyramidal neurons. Neurocan has been shown to inhibit Sema3F-mediated spine retraction in cortical neurons in culture by binding NrCAM and inhibiting its function within the Sema3F holoreceptor ( Figure 4B; Mohan et al., 2018). Neurocan also binds to the Ig2 domain of NCAM at the EphA3 binding site, inhibiting the repellant response to EphrinA5 exhibited by remodeling basket cell terminals (Sullivan et al., 2018).
Another prominent CSPG expressed in brain is brevican. Like neurocan, brevican is involved in synaptic plasticity. In mouse studies examining learning and memory, brevican levels decreased at the beginning of the learning phase and increased after learning was established (Niekisch et al., 2019). Another study found that brevican was essential for the maturation of inputs from excitatory neurons onto PV+ interneuron soma (Favuzzi et al., 2017). Brevican deletion or knockdown in PV+ interneurons causes deficits in spatial working memory and short-term memory, but not long-term memory. Additionally, brevican null mutant mice show longer action potential transmission delays (Blosa et al., 2015). In brevican null mice, PNNs appear intact suggesting that the primary role of this CSPG may be to establish and maintain synaptic function.
ROLE OF CSPG IN RECOVERY AFTER CNS INJURY
A promising future direction of research into PNN function is CNS injury and repair. Using chABC and its ability to restore juvenile connectivity conditions in treated tissue, the enzyme has been employed as a possible therapeutic option for CNS injuries in animal models ( Figure 4C). Following injury to the CNS, a scar forms that consists of glial cell infiltration and an increase in PNN molecules, including CSPGs. This scar grows more quickly than recovery of injured neurons, and blocks neurite growth (Griffin and Bradke, 2020). Digestion with chABC may be an effective treatment for the restoration of neuronal connectivity after such injury. After an acute cervical dorsal column crush lesion, adult rats were able to regenerate a limited number of sensory and cortical spinal tract motor axons and restore both sensory and motor control over limbs affected by the injury after treatment with chABC (Bradbury et al., 2002). Another study showed pericontusional axon sprouting increases in chABC treated rats after a controlled cortical impact injury (Harris et al., 2010). Neurocan has been shown to inhibit the growth of axons in a brain injury model and, along with other CSPGs, is upregulated in injured brains (Asher et al., 2000). Upregulation can be a response to increased levels of transforming growth factor beta (TGF-β) and epidermal growth factor. Recent advances in the deployment of the chABC enzyme show promise. By targeting chABC to axons, neurite extension in SH-SY5Y neurons was significantly increased, even over cells that were transfected with a non-targeted variant of the enzyme (Day et al., 2020). Despite the successes in animal models, no clinical trials using the enzyme have been reported. Innovations with enzyme presentation and continued research with PNNs may allow for a reliable and effective option to be used to restore neuronal connectivity in patients with CNS injuries.
DISCUSSION
The synapse is regulated, strengthened, established, and removed through a host of molecules orchestrated to achieve an optimal functional level of connectivity among excitatory and inhibitory synapses. Synaptic disorders including autism spectrum disorders, schizophrenia, Fragile X syndrome, or intellectual disability often feature disruptions in the function of synapse regulating proteins such as Ig-CAMs, Ankyrins, or CSPGs. The L1 family of Ig-class CAMs are important as obligate subunits of Semaphorin-3 holoreceptor complexes, which mediate developmental spine pruning. All L1-CAMs recruit the spectrin-actin adaptor protein Ankyrin, a high confidence autism risk factor that is critical for both synaptic remodeling and stabilization. At inhibitory synapses, EphrinA ligands and EphA receptor tyrosine kinases function through binding to neural adhesion molecule NCAM to limit the number of perisomatic synapses of basket interneurons, important for both working memory and sociability. The PNN also participates in regulating synaptic plasticity in the developing brain. As the matrix is built, axonal contacts to both dendritic spines and perisomatic regions of pyramidal cells and interneurons are reinforced, plasticity characteristic of juvenile neurons decreases, and new opportunities for connections are blocked. By considering all of these molecular mechanisms as a comprehensive system, new therapeutic targets meant to ameliorate cognitive dysfunction may become available.
AUTHOR CONTRIBUTIONS
BD organized the manuscript. All authors contributed to the writing. | 2021-02-02T17:39:11.898Z | 2021-01-28T00:00:00.000 | {
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144836173 | pes2o/s2orc | v3-fos-license | Content Analysis as a Method to Assess Online Discussions for Learning
One of the challenges for instructors in online education is to create learning opportunities through online text-based discourse. The goal of this study was to examine the use of content analysis to better understand graduate students’ learning in online discussions. The discussion transcripts of a hybrid graduate course were analyzed to determine levels and frequencies of learning or cognitive presence. The analysis of the online discussions was guided by social constructivist rationale, and includes descriptive statistics and inter-rater reliability measurements. Findings show that cognitive presence levels were concentrated in categories marked for exploration and integration. Non-cognitive messages resulted in the highest frequencies of discussion messages, demonstrating indications of social presence and teaching presence. Training was found to be a key factor in resolving coding inconsistencies to improve the reliability of the content analysis. The processes of content analysis applied in this study to evaluate learning in online discussions provided useful information for the development and study of online discussions for learning.
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Article
Online education continues to grow. Over the past decade, increasing numbers of students in the United States have enrolled in online college courses. In 2011, approximately 6,700,000, or 32%, of all higher education students in the United States had enrolled in one or more online courses (Allen & Seaman, 2013). The literature suggests that online education will continue to expand throughout the upcoming decade, although not without concerns. Even though higher education administrators and faculty seemingly have become more accepting of online courses as an extension to the physical campus (Bowen, 2013), chief academic officers of universities perceive that the majority of university faculty members remain skeptical of teaching online (Allen & Seaman, 2013). Many faculty have concerns about the integrity and quality of online courses (Jaggars & Bailey, 2010), although the literature suggests that learning outcomes in hybrid courses are the same and in some respects superior to learning outcomes in face-to-face courses (Means, Toyama, Murphy, Bakia, & Jones, 2010). More research of online discussions across the various disciplines would be helpful those interested in applying asynchronous discussions in their courses. In this research, an instructor of a hybrid course applied an empirical method to assess evidence of learning in the course's online discussions. Faculty who want to substantiate the learning in their courses' asynchronous discussions may want to consider applying similar methods described in this study, and consider publishing the results.
Research on the use of asynchronous discussions used in courses for learning is relatively new, considering that the Internet has been available in schools for only a few decades. In 2003, approximately 93% of the public school classrooms in the United States had Internet access, which is in stark contrast with less than 5% of classrooms having Internet access in 1993 (Parsad, Jones, & Greene, 2005). Over the past decade, there has been an increase in the use of asynchronous discussions as an instructional strategy in faceto-face courses as well as in hybrid and online courses. More recently, strategies such as "flipping a classroom" have increasingly involved the use of online discussions for learning in face-to-face courses to extend instruction outside the physical classroom in preparation for learning inside the classroom (Bergmann & Sams, 2012).
In this study, content analysis was applied to examine the discussion transcripts for learning with respect to a conceptual framework developed from a 4-year study of computer conferencing . The goal was to gain a broader, deeper understanding of the levels of learning in the discussions. An assumption of this study is that online discussions are essential elements of 559019S GOXXX10.1177 1 California State University, Sacramento, USA online learning. Throughout this article, the terms online discussions or asynchronous discussion are used interchangeably and are intended to refer to time-delayed discussions, not synchronous real-time discussions.
Literature Review
Learning theories provide a basis for understanding how learning occurs in online discussions. Principles of learning theories are used to explain or support teaching in meeting the learning needs of students (Borich & Tombari, 1997;Dillenbourg, 1999;Hofer, 2001). Particularly relevant to online learning are the principles of constructivist learning theories, which include collaboration, interaction, and the use of language (Bruner, 1986;John-Steiner & Mahn, 1996;Vygotsky, 1980). In turn, these constructivist principles fundamentally characterize how learning occurs in asynchronous discussions (Lebow, 1993;McLoughlin & Oliver, 1998;Swan, 2005;Yuan & Kim, 2014) and serve as the basis underlying the approach to this study in examining the discussion transcripts of graduate students for evidence of learning.
Choosing discussion strategies is an important step in the planning of online learning. A review of literature identifies factors to consider in planning for online discussions, which may help planning for the discussions.
Online Discussion Strategies
Asynchronous discussions are typically an important part of learning in online or hybrid courses. Discussions offer a way for students to learn and articulate their understanding of learning through interactions with each other and the instructor (Parker & Hess, 2001). Darabi, Liang, Suryavanshi, and Yurekli (2013), in their meta-analysis of 80 studies of online discussions, found that "learners responded better to strategic and productive discussion than when they were asked to elaborate on a topic" (p. 239). Although the purpose of this study does not involve an in-depth analysis of the various online discussion strategies, it does give an overview of strategies and factors that are relevant in planning. Further studies that describe the processes of choosing and applying text-based discussion strategies would be helpful.
The Socratic method of discussion has traditionally been recognized as an effective way to help students learn beyond memorizing and regurgitating facts (Hansen, 1988). Socratic discussion methods employ thought-provoking questions which are evident in online discussion strategies such as in online debates and case studies (Yang, Newby, & Bill, 2005). Literature circles are another Socratic method of discussion that has been used effectively to support learning in both online and face-to-face discussions. Literature circles begin with a discussion of a text, and are guided by open-ended questions that are designed to promote deeper thinking and discussion of the text. Having literature circles online provides for added reflection opportunities for students in reading the transcripts of the discussions, which supports broader and deeper perspectives and understanding of the discussion topic (Larson, 2009). The think-pair-share discussion method has been structured effectively for use in online discussions (Johnson & Aragon, 2003). In this strategy, students first think independently about a problem or a question, are then paired or grouped to discuss their thoughts, ending with the whole class sharing and discussing the topic. The think-pair-share discussion strategy builds a strong foundation for communicating ideas with a range of fellow students and the instructor (King, 1993).
In a study by R. S. Anderson, Goode, Mitchell, and Thompson (2013), they examined a group of doctoral students' perceptions of four different online constructivist discussion strategies, including problem-based learning (PBL), discussion web strategy, 3-2-1 strategy, and case study. Each of the strategies was said to reflect social constructivist learning because of the interaction involved in the discussions and the development of personal meanings associated with the discussions. In the PBL discussion strategy, the students discussed self-directed problem-solving objectives. In the discussion web strategy, the students identified and described the pros and cons of a particular topic, justifying their position. In the 3-2-1 online discussion strategy, the students read a text, selected three key points from the text, provided two supportive explanations, and then posed one question related to the reading. In the case study strategy, the students analyzed real-life situations, read case information, made reflective connections, stated opinions, and asked and responded to questions. Students' perceptions of these discussion strategies were collected through surveys and interviews, and were analyzed, coded, and categorized by four coders based on a process of inductive reasoning. The results showed that the students believed all of the discussion strategies to be effective for particular types of discussions.
Factors in Planning Online Discussions
Consideration of the practical aspects or factors of online discussions can be helpful in planning for the use of them in online learning. Among the factors to consider are time, student attitudes, and technology.
Asynchronous discussions require reading, writing, and comprehension tasks that arise from students needing to develop, post, and read text-based communication. This can result in more time required of the students to complete online discussions compared with the amount of time typically spent in face-to-face class discussions (Meyer, 2003). Instructors would be wise to consider the coordination needs in scheduling the time for students to read and post to the discussions. Reading, writing, and the increased amount of time that students spend in text-based discussions can provide for more focused and deeper learning through the increased opportunities to read, reflect, and respond (Benbunan-Fich & Hiltz, 1999;Bliuc, Ellis, Goodyear, & Piggott, 2011;Maor, 2003).
Asynchronous text-based discussions may have a positive impact on student attitudes toward discussions by lessening any difficulties arising from students who are reluctant to participate, feel confronted in the discussions, or are not given enough time to voice their opinions. The atmosphere in asynchronous discussions is seen by some students to be easier to handle, less stressful, equitable, and less likely to be dominated by a few individuals (Wang & Woo, 2007). However, there are disadvantages of text-based discussions that students may perceive negatively. Text by itself does not communicate the nuances of tone of voice nor does it replace visual cues of face-to-face discussions. The use of emoticons in online discussions can suggest tone of voice that may clarify the meaning behind the text (Tiene, 2000). More studies are needed to understand the effect and practice of using emoticons in online academic discussions.
Students need to possess functional technical skills to participate in asynchronous discussions. The necessary level of technical difficulty is not extremely high for most students, but a lack of familiarity with the online discussion features may cause frustration among some students. Frustration with technical difficulties may affect the quality of participation in the discussions (Davidson-Shivers, Muilenburg, & Tanner, 2001). The knowledge and skills necessary for students to participate in asynchronous discussions can be effectively addressed in instruction.
Reviewing various discussion strategies and considering factors that can affect asynchronous discussions will aid in planning. Analyzing the discussions is an additional task that can provide information for reflecting and revising the overall discussion strategy in efforts to understand and improve the online discussion's effectiveness for learning.
Methods to Analyze Online Discussions
There are many analysis methods reported in the literature that have been used to assess learning in online discussions, few have been applied extensively. Among the analysis methods, two methods appeared to have been applied in several other studies, and were considered for this study, the Interaction Analysis Model (Lucas, Gunawardena, & Moreira, 2014) and the Community of Inquiry (COI) framework (Garrison & Arbaugh, 2007).
A study that used the Interaction Analysis Model from Gunawardena, Lowe, and Anderson (1997) described an analysis of an online debate. Gunawardena et al. characterized online debate as a " . . . co-creation of knowledge and negotiation of meaning" (p. 406). The Interaction Analysis Model consisted of five categories or phases, including (a) sharing or comparing information, (b) discovery and exploration, (c) negotiating of meaning or co-construction of knowledge, (d) testing and modification or synthesis, and (e) agreeing or application. From the coding and analysis, based on these five categories, much of the participants' discussions were determined to be characteristic of two categories, exploration or discovery, and negotiation of meaning. The Interaction Analysis Model categories have some similarities of categories with Garrison's COI framework's practical inquiry model. However, the COI framework, which was chosen for this study, appeared more broad-based, including categories of teaching presence, social presence, and cognitive presence.
Theoretical Rationale
The theoretical framework of this study is based on the COI framework , which is grounded in constructivist learning theory that considers collaboration, reflection, and critical analysis as essential to learning. The COI framework is comprised of three core elements: social presence, teaching presence, and cognitive presence. Social presence, teaching presence, and cognitive presence are all intertwined. "Cognitive presence is defined as the extent to which learners are able to construct and confirm meaning through sustained reflection and discourse" (p. 11), and can be used as a " . . . means to assess the nature and quality of critical, reflective discourse that occurs within the text based educational environment" (p. 7).
The element of cognitive presence was selected as a primary focus for this study based on reviews of research studies that employed content analysis to assess online discussions. Many of the content analysis studies that were reviewed were identified in articles from De Wever, Schellens, Valcke, and Van Keer (2006) and Rourke, Anderson, Garrison, and Archer (2001). Content analysis is a qualitative research method that has been used fairly extensively, and its selection followed a "directed approach," where the analysis method is based on methods identified in the literature (Hsieh & Shannon, 2005). In research pertaining to the content analysis of asynchronous discussions, research literature highlights the importance of reporting the basis of its theoretical foundation including information regarding the unit of analysis and the reliability of the study (De Wever et al., 2006). The theoretical rationale, unit of analysis, and reliability of analysis are all reported in this study.
In developing the COI framework, a practical inquiry model was developed. The practical inquiry model was designed to be applied to analyze transcripts of online discussions for cognitive presence. The practical inquiry model consists of four phases grounded in perception, deliberation, conception, and action. Each phase reflects a process leading to problem resolution beginning with identifying or understanding the problem, termed the triggering event. The second phase involves exploration of the topic. In the third phase, integration, possible resolutions or solutions or conclusions are identified, and the fourth phase, resolution, is where the solutions or conclusions are selected and applied. These four phases or categories (triggering event, exploration, integration, and resolution) represent a process of evolving learning in asynchronous discussions that can be supported and analyzed. The process to identify evidence of each of the four phases is an interpretive process. To guide the process of identifying cognitive presence in online discussions, categories of descriptors, indicators, sociocognitive processes, and examples act as guidelines to facilitate content analysis coding of the discussion transcripts . According to Garrison et al. (2001), it is important to note that the practical inquiry model indicators should "not be seen as immutable" (p. 9), meaning that other studies using the practical inquiry model may find a need to refine or revise the criteria to meet specific analysis needs, as was the case for this study.
Method
In this study, an empirical method, content analysis, was chosen to assess cognitive presence of three asynchronous discussions of graduate students in one of their courses. "Content analysis is a research technique for making replicable and valid inferences from texts (or other meaningful matter) to the contexts of their use" (Krippendorff, 2012, p. 24). Content analysis enables a process to systematically examine the quality of learning in online discussions (Gunawardena et al., 1997). Although the use of content analysis dates back to the 1940s and 1950s, it was not until the 1980s and 1990s that it began to be more frequently applied to study learning in asynchronous discussions. Henri (1992) described computer conferencing as a "gold mine of information" (p. 118) that would provide researchers a rich resource to analyze and advance online learning. Use of content analysis to assess online discussions has increased over the past 20 years, just as Henri had predicted, but concerns about lack of uniformity and disclosure of the analysis methods have arisen (De Wever et al., 2006;Rourke et al., 2001).
Issues in comparing content analysis studies of online discussions have arisen due to a lack of consistency in the different analysis instruments used (Rourke & Anderson, 2004). "This lack of replication (i.e., of successful applications of other researchers' coding schemes) should be regarded as a serious problem" (Rourke et al., 2001, p. 6). Consequently, research literature has stressed the need for more studies to employ similar instruments (T. Anderson, 2005;De Wever et al., 2006), which in turn should increase the reliability and validity of these types of studies (Stacey & Gerbic, 2003). The importance of building on previous research influenced choosing the method and the coding instrument of this study. Repeating research designs helps establish the reliability of the results, which can be obtained from repeated use of the same instrument. Further information regarding this practical inquiry model, which has been applied in other content analysis studies of online discussions, can be found in studies from Akyol and Garrison (2011), de Leng, Dolmans, Jöbsis, Muijtjens, and van der Vleuten (2009), De Wever et al. (2006), Fahy (2005), and others.
Analysis of the results of this study is aimed to (a) aid the researcher in the planning of future asynchronous discussions in the graduate program, (b) provide information for instructors who are interested in studying the learning effectiveness of their asynchronous discussions, and (c) apply lessons learned from this study to further studies involving the use of content analysis as a method to evaluate online discussions. One goal is to continue using content analysis to better understand the use of this method to assess online learning strategies. Analysis of other discussions from the same graduate cohort of this study is planned.
Research Questions
Research Question 1: What were the levels of cognitive and non-cognitive presence in a cohort of graduate students' online discussion transcripts from one of their college courses? Research Question 2: Considering that the use of roles in online discussion was the main topic in the first online discussion in the course, what were the students' perceptions at the end of the semester regarding the use of roles in online discussions?
In the "Discussion" section of this study, the researcher reflects on the use of content analysis and the practical inquiry model as a means to assess the learning effectiveness of online discussions.
Participants and Context
In this study, three online discussions of a hybrid graduate course, the "Fundamentals of Online Pedagogy," were examined for cognitive presence. The researcher of this study was the instructor of the course, which was primarily online, but included three face-to-face class sessions at the beginning, the middle, and the end of the semester. The course is part of a master's program designed to be completed entirely through a hybrid delivery system over four consecutive semesters. All of the courses in the students' graduate program typically met face-to-face only 2 to 3 times each semester. The course examined for this study was situated in the first semester of the students' graduate program. There were a total of 19 students (10 females, 9 males) participating in the course, although only 15 students finished the course. A random sample size of students (N = 15) was chosen for the analysis. Three separate discussions were analyzed out of eight total online discussions in the course. Each of these discussions included three discussion groups, except the first discussion which had five groups, and each of the discussions occurred over a week time period. The reasoning behind selecting and analyzing the first three online discussions of the course was to allow for the opportunity to make observations of the students' learning progression in the course from the beginning of the semester. Studies of the other discussions in the same course and same students will be based on the results of this study.
Online Discussion Strategy
The online discussion strategy used in each of the three discussions of the study was identical, consisting of a format similar to the 3-2-1 discussion strategy, which included reading a text, finding key points in the text, providing supportive explanations, and posing questions. The instructions for the discussion included several items: • • Students were divided into groups for the online discussions. • • Each discussion required reading of an initial journal article. • • Groups were to identify and discuss three main points from the article with a goal of discussing and identifying related topics to broaden and deepen students' understanding of the topic. • • Students were then to search for another article based on the three main points that had been identified and discussed; read the new article; and then describe, discuss, and synthesize the concepts from the two articles. • • To conclude the discussions, summaries were posed synthesizing each group's discussions. • • Role assignments were assigned to each member of the discussion group, which included monitor, encourager, facilitator, quality assurance checker, and summarizer.
Data Collection
The analysis of the online discussions was unobtrusive, occurring after the students had already completed the course and been given their final grades. Technology was used to collect, process, and organize the data for analysis. Blackboard, the University's learning management system, was used to archive the discussion transcripts. Learning management systems, such as Blackboard, support the use of content analysis to evaluate online discussions with their capacities to store online discussion transcripts that are easily accessible for later analysis. The discussion transcripts for this study were exported from Blackboard, and then imported into HyperRESEARCH, a qualitative software program that was used to organize and code the discussion transcripts.
A secondary data source included student survey data which were collected at the end of the semester. The survey inquired of the students about their perceptions regarding the use of roles in online asynchronous discussions, and was the subject of Discussion 1, which was based on the reading of an article on the use of roles in online discussions from De Wever, Van Keer, Schellens, and Valcke (2010).
Coding
Two coders, the researcher, who was the instructor of the course, and another university instructor coded the three sets of discussion transcripts. Training was provided to the coders to aid them in coding the discussion transcripts. The training involved the researcher/instructor of the course describing and demonstrating to the second coder how to apply the coding instrument (Table 1) to the discussion transcripts. Included in the training were discussions between the two coders regarding the instructions for Discussion 1 that had been given to the students who participated in the discussion. The training occurred during 1 week, totaling approximately 3 to 4 hr. Discussion transcripts from Discussion 1 were used in the training to demonstrate the coding. Following the training, the coders worked independently in coding the units of analysis.
The units of analysis consisted of each message thread from each of the discussion participants in the three discussions. Clear demarcation at the beginning of each message identified for the coders where to begin and end each coding effort. Each of the messages was analyzed by the two coders and classified according to the indicator representing its level of cognitive presence (see Table 1), which was based on a modified version of the practical inquiry model . During the coding, if the coders determined that more than one indicator of cognitive presence was evident in a message thread, the indicator selected was to be based on a preponderance of evidence in the message. Training for the coders was considered a critically important factor that could affect the reliability of the content analysis applied in this study.
Variables
The variables related to cognitive presence were defined based on four categories of Garrison's practical inquiry model , including triggering events, exploration, integration, and resolution. Each broad category contained a sub-level of indicators, which were used as the basis to code each discussion message. Although the sublevel indicators were drawn from Garrison's practical inquiry model, they were modified to facilitate the coders in identifying evidence in the online discussion transcripts of this study. Categorization of the sub-level indicators provided deeper insight into the cognitive and non-cognitive presence displayed by the students in the discussions. Table 1 shows the sub-level indicators for the broad categories of cognitive and non-cognitive presence, including the percentages of occurrence for the three discussions. The percentages of messages identified for each indicator reflected in Table 1 represent an agreed-on coding of each message based on discussion and reconciliation of messages between the two coders after all of the discussion messages had been coded.
The discussion messages were separated for each cognitive and non-cognitive broad category by group for each of the three discussions (see Tables 2, 3, and 4). These three tables show the number of messages coded for each broad category, including the triggering event, exploration, integration, resolution, and non-cognitive. Inter-rater reliability of the coding was calculated for Discussions 2 and 3 with the use of Cohen's kappa statistical measurements, including percentages of agreement between coders (see Table 5).
Potential for Coding Error and Bias
The researcher of this study was the instructor of the course with 20 years of experience in the field of educational technology. Steps were taken to reduce any ambiguity and bias in the coding process: (a) Student names were removed from the discussion transcripts in preparation for the coding. This eliminated coder bias toward any student names. (b) Training and practice coding of Discussion 1 was provided to emphasize procedures for the coding based strictly on the criteria established for each indicator (see Table 1). Training was considered essential to reduce ambiguity in the judgment process for coding each message. (c) Demarcation of the unit of analysis of the coding was discussed between the coders to eliminate any errors from confusion in the unit of data to code. The unit of analysis for coding consisted of the message as opposed to sentences or paragraphs. However, there was the possibility of messages containing evidence of more than one indicator, which was addressed in this study by coding the messages based on the preponderance of evidence in the message.
Results
A total of 724 messages were posted in the three discussions. Discussion 1 included a total of 233 messages, Discussion 2 contained 200 messages, and Discussion 3 had 291 messages. For all of the three discussions, the average percentage of messages coded for each cognitive presence category resulted in 6.8% triggering events, 29.4% exploration, 23.8% integration, and 2.9% resolution. In addition, messages coded as non-cognitive accounted for 37.1% of the total messages. Three categories, non-cognitive, exploration, and integration, accounted for roughly 90% of the total messages in the three discussions. The lowest percentage of responses was in the categories of resolution (2.9%) and the triggering event category (6.8%), which were both considerably lower than any of the other three categories. The messages categorized as triggering events primarily consisted of descriptions of the articles assigned by the instructor for the discussions. The discussion messages categorized as resolution primarily consisted of student summaries of the discussions with little back-and-forth discussion among the students about the summaries. In online discussions, students appear to need more direct guidance to increase discussion that would be characteristic of resolution.
Messages coded as exploration (29.4%) and integration (23.8%) accounted for the highest frequencies of cognitive presence in this study. High frequencies of exploration and integration messages have been similarly reported in the findings of other studies that also relied on the primary inquiry model and content analysis to evaluate learning in the discussions Liu & Yang, 2012). The non-cognitive category accounted for the overall highest frequency (37.1%) of messages, which was 7.7% higher than the next highest category of exploration (29.4%). Students who did not follow the instructions in Discussion 1 appeared to directly influence the higher frequency of messages coded as non-cognitive (47.1%), which was substantially higher than non-cognitive messages in Discussions 2 (27.5%) and 3 (35.5%). Discussions 2 and 3 did not have as many messages asking questions about the instructions and procedures of the online discussion. A solution to reduce potential non-cognitive messages is to teach the students about the procedures and processes prior to the first discussion. In a fully online course, this could be accomplished through a separate discussion thread that focused exclusively on protocols of the discussions. In a hybrid course, this could be addressed in the first face-to-face class. Displayed in Tables 2, 3, and 4 are the total number of messages for each of the three discussions which are listed by group showing the total percentages of cognitive presence (triggering event, exploration, integration, and resolution) and non-cognitive presence. There were five groups participating in Discussion 1, three groups in Discussion 2, and three groups in Discussion 3. A pattern appeared to be emerging from the overall frequency of messages coded for the different categories of cognitive and non-cognitive presence. Messages coded as non-cognitive or exploration were usually the most frequent type of messages identified in each of the three discussions. Messages coded as resolution or as triggering events were identified as the least frequent messages in all three discussions. Messages coded as integration were usually identified as the third most frequently occurring messages in the three discussions, except in Discussion 3 where they were second highest, only 3.2% lower than total non-cognitive messages.
Discussion 1 Cognitive and Non-Cognitive Presence
Discussion 1 had the highest percentage of messages coded as non-cognitive, 47.6% (see Table 2) of the messages, compared with an average of 37.1% of non-cognitive messages for all three discussions. Table 1 shows that most of the messages classified as non-cognitive were coded for clarifying discussion procedures. Messages coded as exploration were slightly higher at 30.5%, when compared with an average of 29.4% for all three discussions. Integration messages in Discussion 1 were lower at 18%, similar to Discussion 2's, but considerably lower than Discussions 3's percentage of messages categorized for integration at 32.3%. Few messages (0.9%) were coded as triggering events. However, some of the messages that were coded as exploration and integration contained evidence characteristic of triggering event criteria, but were coded as exploration or integration due to the preponderance of evidence in the message. Another factor affecting the low frequency of triggering events in Discussion 1 was partially a result of the students being introduced to the topic of Discussion 1 during the first faceto-face class meeting. Students having the opportunity to talk to each other about Discussion 1's reading assignment in a face-to-face environment eliminated some of the online discussion that likely would have occurred.
Discussion 2 Cognitive and Non-Cognitive Presence
In Discussion 2 (see Table 3), the data show an increase of 10.6% in messages coded as triggering events, up 11.5% compared with Discussion 1 at 0.9%, while the average for triggering events for all three discussions was at 6.8%. The content of the triggering event messages in Discussion 2 clearly showed that the students were able to adequately identify and describe the topic of the discussion, which was based on an article focused on social presence in asynchronous discussions.
Also in Discussion 2, students were much more active exploring and discussing additional perspectives related to the topic of the assigned reading. The increased discussion activity resulted in the highest percentage of messages coded for exploration with 41%, compared with the 29.4% exploration average of all three discussions. Furthermore, data for Discussion 2, as seen in Table 1, show that 14% of the students' messages focused on asking questions seeking specialized information. Discussion 2 also showed a 20.1% decrease of messages coded as non-cognitive compared with Discussion 1, due to fewer messages coded for clarifying discussion procedures. It appeared that the students had become more comfortable with the procedures for the online discussion. Also, in Discussion 2, messages identified as resolution remained low at 2% of the total messages, which consisted of students reporting a summary that synthesized the overall discussion, although there continued to be limited discussion among the students regarding the summary.
Discussion 3 Cognitive and Non-Cognitive Presence
Discussion 3 had the highest frequency of messages coded for integration at 32.3%, compared with an average of 23.8% for all three discussions. Integration reflects a higher level of thinking skills reflecting students' ability to synthesize the discussion concepts and form summary conclusions. In Discussion 3, there was a higher percent of non-cognitive messages at 35.4%, compared with 27.5% in Discussion 2. The increase of non-cognitive messages in Discussion 3 from Discussion 2 was due to a moderate increase in messages that were seeking clarification (see Table 1). Discussion 3 was focused on "concept mapping," which was for some students a difficult concept to read about and discuss and then apply. In the course, there was an assignment that required the creation of concept maps. Some confusion arose among the students between the assignments requiring the actual building of a concept map with the online discussion about concept mapping. Conversely, the increase in integration messages appeared to be a result of more discussion requiring the forming of solutions of how to develop concept maps that would be applied.
Reliability
Inter-rater reliability identifies the extent of agreement among the coders and takes into account any agreement occurring by chance. Information of the inter-rater reliability provides an indication of the reproducibility and stability of a study, and is considered an essential element of content analysis (De Wever et al., 2006;Lombard, Snyder-Duch, & Bracken, 2002).
There are differences in interpretations regarding acceptable levels of inter-rater reliability coefficients. According to Landis and Koch (1977), kappa coefficients of "0-.20 are slight, .21-.40 fair, .41-.60 moderate, .61-.80 substantial, and .81-1.00 perfect" (p. 165). However, literature also shows the existence of differing interpretations regarding coefficient levels, including interpretations saying that coefficients below .8 give rise to concern (Lombard et al., 2002). In a similar study, Garrison et al. (2001) assessed cognitive presence in two online discussions and calculated kappa coefficients at levels of . 35, .49, and .74, and .45, .65, and .84. The increasingly higher coefficients, from .35 to .75, and from .45 to 84, were thought to be a result of increased training for the coders. Training appeared to be an influencing factor.
In content analysis, latent content is often considered a challenging area of concern. The latent nature of discussions involves potential reliability issues related to the coders' " . . . interpretations of the meaning of the content" (Potter & Levine-Donnerstein, 1999, p. 259). To alleviate any issues of reliability associated with latent content, training proves to be a key factor. Higher coefficients are thought to be more achievable as a result of coders having more training with the processes related to coding (Riffe, Lacy, & Fico, 1998).
In Table 5, the kappa coefficients and percentages are displayed for Discussions 2 and 3. Discussion 1 is not displayed as it was used in the training process for the two coders. There were three separate group discussions in Discussion 2 and three group discussions for Discussion 3. Inter-rater reliability coefficients were calculated for each group discussion. Most of the coefficients were in the moderate range from .41 to .60. However, for Discussion 2, Group 2, an inter-rater reliability of .85 was calculated, which is a substantially higher kappa coefficient than all of the other discussions. The coders periodically compared and discussed their coding of Discussion 1. For Discussions 2 and 3, the coders compared and discussed their coding only after the entire coding was completed, and did not periodically compare coding results during the coding of the discussion transcripts.
The training that was provided to the coders of this study included (a) discussions of the coding instrument and the indicators, (b) discussions on how to apply and record the coding, (c) a review of the instructions and procedures for the discussions, and (d) practice coding of Discussion 1, including discussing the differences in the coding results and arriving at an agreement to resolve differences.
The low reliability coefficients were a result of differing interpretations between the two coders regarding the content of the reading assignments. These differences were examined, discussed, and resolved after the coding of Discussions 2 and 3. The sub-level indicators appeared to be a source of the problem in applying and coding the discussion messages. In discussing the differences of the coding, each message was considered in relation to the sub-level indicator and in relation to the broader category (triggering event, exploration, integration, and resolution).
Descriptions of the type of content in a message would meet specific categorization criteria based on the sub-level indicators of each broad category. Messages coded as triggering event were to basically describe the initial article. Differing scores arose from the coder not knowing the difference between new information from a new article from information that was derived from the assigned article of the discussion. Thorough review of the assigned article was needed.
Sub-level indictors for integration and exploration were the areas where many other scores differed. Examination of differences of integration and exploration made apparent a need to further refine the sub-level indicators along with an examination of the prompts or instructions for the discussion. Each of the messages coded differently in these two areas were discussed in relation to the articles associated with the discussions until an agreement was made.
As a result, specific type of training was also identified that would need to focus on the subject matter of the topics that were the focus of each online discussion. Overall, the process of evaluating the coding provided insight into refinements or changes to the discussion strategy that may have a positive impact on the use of online discussions for learning.
Survey Results
Among the learning goals for the students in the course involved in this study was to develop knowledge of online discussion strategies. A survey was given to the students at the end of the semester to examine their perceptions of a discussion strategy that was the main topic of Discussion 1, which was focused on the use of roles in online discussion.
The survey results collected at the end of the semester showed that all of the students, except one, believed that the use of roles in online discussions would result in generating greater participation from all the members of the discussions, which is a contributing factor for quality and productivity in online discussions. The student who did not wholeheartedly support the use of roles as essential to online discussions believed that roles required too much structure in an online discussion, which could inhibit free-flowing discourse. Second, the role of summarizer was identified by a majority of students as the most important and difficult role in an online discussion. Students also indicated in the survey that they had difficulty with summarizing skills, although they did believe that summarizing discussions would help foster deeper understanding of the topics being discussed. In addition, students were evenly divided in their beliefs about rotating roles in discussions. Half of the students believed that maintaining the same role was more beneficial to the discussions than changing roles from discussion to discussion. Maintaining the same role was believed to help the students master the role, which would lead to higher quality discussions. The other half believed that changing roles from discussion to discussion would help learning new skills from roles that may not have been chosen. Most students believed that their choice of roles would be influenced by their comfort level, although the students also indicated that there was value in being forced out of their comfort zone. Overall, there was a strong indication that the students favored the use of roles as an important online discussion strategy for learning.
Discussion
Examining the three discussions through the research framework of this study provided deeper insights into the impact of online discussion strategies in learning. Reviewing the literature for online discussion strategies revealed a need for more perspectives of research-based reviews of online discussion strategies. For example, Darabi et al. (2013), in their meta-analysis of online discussion strategies, could only locate eight articles that met their rigorous research criteria. In determining a theoretical rationale to frame this study of online discussions, also revealed was a dearth of frameworks that had been applied extensively in research. However, the practical inquiry of Garrison et al. (2001) that was applied in this study appeared to be flexible and adaptable enough to serve as a basis to analyze many types of online discussions. Applying the practical inquiry framework to analyze the three online discussions of this study made evident the need for careful instructional planning of the online discussion prompts with consideration of triggering events, exploration, integration, and resolution.
Overall, Tables 2, 3, and 4 show strong evidence of cognitive presence in the three online discussions. The distribution of discussion frequencies was acceptable with a substantial volume of the discussion posts evident of exploration and integration. The small percentage of triggering event messages was acceptable with 8% to 10% of the messages adequately covering the necessary information essential to the discussions. Students appeared to progress fairly well through the discussions, first identifying the topic of discussion (triggering event), expanding with ideas for further discussion (exploration), and then discussing and integrating new ideas. Strengthening the discussions strategy could be accomplished with more direction in applying solutions or conclusions to real or virtual situations. In the original instructions for the online discussions given to the students, there were not any directions instructing the students to apply their conclusions to real-life or virtual settings, although there were a few students who were K-12 teachers that did discuss applications of the conclusion to their real-life classroom situations.
Non-Cognitive Presence
The percentages of non-cognitive messages appeared rather high in the first discussion, went down in the second discussion, and then increased slightly in the third discussion.
Online discussions are sometimes filled with non-cognitive off-task messages, which was not a problem in this study. Non-cognitive messages that seek clarifications of the discussion instructions can help facilitate the discussion, and can be supported in various ways depending on whether the course is a hybrid or fully online course. Clarification of instructions can be taught in face-to-face class sessions, or separate threads dedicated to clarifying instructions for the discussion would help. "Teaching presence," which is a major concept of the COI , can be elevated in online discussions through separate discussion threads led by the instructor of the course. Students appear to expect the instructor to have strong teaching presence in online discussions.
The messages coded as encouraging are characteristic of social presence, also a major concept of the COI model. Messages coded as encouraging were moderately evident throughout the three discussions. Encouraging messages can increase social presence in online discussions, which promotes positive outcomes. Students in this study were influenced by the use of encourager roles, which was used in all three discussions. The use of roles as a discussion strategy has been shown to have a positive impact on student interaction in asynchronous discussions (De Wever et al., 2010;Yeh, 2010), which was confirmed by this study.
Reflections on Content Analysis and the Practical Inquiry Model
T. Anderson (2005) suggested that there must be an easier way to support and confirm acceptance of online learning other than through content analysis, and that few of the content analysis methods for online discussions have been used repeatedly enough, creating a problem comparing studies. Although difficulties do arise in applying content analysis to study online discussions, for those desiring to improve their online discussions, the benefits of applying content analysis outweigh the difficulties in its use.
Major difficulties in using content analysis include (a) modifying the indicators' criteria, (b) developing training for the coders, (c) training coders, and (d) the time it takes to complete these tasks. Content analysis is a tedious process to apply in the analysis of online discussion transcripts. Thorough training is required for the coders to establish acceptable reliability measurements. There has been some difficulty in training others to discern the differences between phases of the practical inquiry model. The integration " . . . phase is the most difficult to detect from a teaching or research perspective" (Garrison et al., 2001, p. 10). The inter-rater reliability of this study was affected by issues that typically affect content analysis studies, relating to the coders being knowledgeable of the subject matter in the study, and crafting measurability instruments that address this issue (Potter & Levine-Donnerstein, 1999).
Limitations
Limitations of this study include a small sample size involving 15 graduate students from an educational technology master's program in their first semester of the program. The results may be generalizable to analogous populations using comparable online discussion strategies. However, the design of coder training and the development of criteria for coding indicators may possibly affect the results. The primary inquiry model, which includes the categories of triggering events, exploration, integration, and resolution, appears to provide a strong framework to serve as a base for the development of the coding criteria. Other factors that may affect generalizability include variables related to student backgrounds (e.g., student context factors including academic, language, social, and socioeconomic factors). These factors should be considered in developing and examining online learning (Swan, 2001).
Other limitations include the following: The study only examined one element, cognitive presence, from the COI model , excluding elements of teaching presence and social presence. Although there were no plans at the onset of the study to examine teaching and social presence, there is some indication of their presence in the non-cognitive messages. Social presence appears to be evident in the messages containing "encouraging" comments, and teaching presence is potentially present in the messages containing comments related to instructional procedures. It is worthwhile to examine these two areas more closely in future studies.
Conclusion
The research literature on content analysis of online discussions calls repeatedly in its conclusions for the need of studies that contain all of the following: (a) a systematic coherence between theory and analysis categories, (b) clear choice of the unit of analysis, and (c) information about the inter-rater reliability procedures (De Wever et al., 2006). These are the essential requirements needed for content analysis studies that provide the means for professional comparisons that may contribute to improved conditions in online teaching and learning. In addition to meeting the above three requirements necessary to compare studies applying content analysis to online discussions, there are other concerns to address in the process of applying content analysis to study online discussions.
There are several important steps in applying methods of content analysis to analyze learning in online discussions. These steps are not meant to be comprehensive but are lessons learned from this study: 1. Use a software program such as HyperResearch or Atlas.ti, which is very helpful in sorting, analyzing, and reporting data.
2. Use an existing theoretical rationale or research design such as the COI . Although there is pressure to develop an individualized instrument, researchers and practitioners can benefit from repetitive studies that thoroughly vet research designs. 3. Examine and understand the online discussion strategies in meeting learning objectives associated with the online discussions. 4. Modify the criteria (indicators) of the coding instrument to align with the identification of the content of the online discussion messages in relation to the learning objectives. 5. Select coders who are knowledgeable with the subject matter of the online discussions. 6. Plan thorough training for the coders. 7. Assess training of coders for inter-rater reliability measurements that are at least .8. 8. In analyzing the online discussions, analyze for cognitive presence, teaching presence, and social presence.
This study used an existing model for content analysis to analyze online discussions of a course previously taught. The purpose was to improve the use of online discussions to support learning in hybrid courses in education. The study confirms the value by applying the framework practical inquiry model and further identifies important steps or lessons learned in applying content analysis to assess learning of online discussions. Finally, it was the experience of the researcher of this study that the process of applying content analysis to examine a course's online discussions will provide additional perspectives on development and use of online discussions for learning.
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 and/or authorship of this article. | 2019-05-05T13:05:43.046Z | 2014-10-01T00:00:00.000 | {
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234444473 | pes2o/s2orc | v3-fos-license | Re-Inscription of Black History/Body in Morrison’s Beloved and Paradise
A bs tr ac t This article contends how Toni Morrison has used her black fiction to reject the dominant conceptions of reality and truth constructed by the white pahllogocentric discourses that tended to perpetuate white power interests. The poststructuralist assumption that knowledge and reality are socially constructed phenomenon provides useful insight into Morrison’s narrative strategies and helps understand how, on one hand, she represents the ways the history of the black Africans had been badly disfigured in the white discourse resulting in the construction of the negative stereotypes of the black people as barbarians, savages, and uncivilized people whose mythical history and social values were invalidated as inauthentic and savage that needed the enlightening intervention of the white Europeans and, on the other hand, apart from revealing the discursive facts that control reality formation, she disrupts and displaces dominant and oppressive white knowledges.
Introduction
Morrison's commitment with rewriting the history of her people, gender, myths and communal identity is evident from the fact that all of her novels, particularly the Beloved (1988) and Paradise (1997) deal with the questions of slavery, survival, repressed desires, silenced voice, misrepresentations of her people, the psychological impacts of their subjection to slavery, and their sustained efforts for freedom and self-actualization. She has taken the responsibility of redressing the wrongs done to her people by the so-called champions of the rights of human beings, by deconstructing the racially biased representations of the black people in a unique ecriture feminine mode of narration. In her fiction, she articulates the traumatic history of the institution of slavery and the terrible after effects of this psycho-physical subjection of the black races. In this regard, Beloved and Paradise, privileging women's experience of traumatizing slavery and its terrible effects upon African subjects, epitomize the dehumanized history of the centuries long suppression of the Black generations, the suppression that alienated them from their past traditions, mythical beliefs, and strong connection with the world of nature. As the subversion of dominant patriarchal order requires resistance to, rather than uncritical acceptance of, the prevalent forms of subjectivity, Morrison's serious engagement with the black feminist writing from the indigenous perspective has led to the retrieval of Afro American people's mythical past and glorious integrated communal life. Establishing a feminine tradition of Afro American writing, the one that subverted the phallogocentric structure of language which is particularly designed to distance woman from her body, and which conversely serves male purposes, she employed forms of language that reframe linguistic structures, push out rules, and uses Kristeva's (1986) quintessential pre-oedipal semiotic language rather than Lacan's (1993) symbolic language. Her use of feminine language that "surpasses the discourse that regulates the phallocentric [discursive] system" (Cixous, 1976, p. 883) to reconstruct the identity/subjectivity of black women is subversive and cutting edge in its non-confirmative stance towards phallogocentric writing and thus signals the great contribution of the Afro American women towards the inclusion of the black women writers in the cannon.
Literature Review
Helen Cixous (2002) encourages women to write since it is as much important for them as it is for males. For Cixous (2002) women need to completely own their bodies and experience because they are the only owner or holder of their bodies, not men. The best way to actualize their potential identity is by writing but women were kept away from the process of writings by patriarchy because the act of writing was supposed to be reserved for "great men". Subverting this myth of writing attached with men she invites women to "Write! Write, let no one hold you back, let nothing stop you: not man" (Cixous, 1976, p. 877). Luce Irigaray (2004) is also one of the writers who talk about women writings by making a critique on the ideas of Sigmund Freud. Luce Irigaray argues that "in our culture, woman is outside representation: 'the feminine has consequently had to be deciphered as forbidden, in between signs, between the realized meanings, between lines" (Irigaray, 1974, p. 20). For that reason, females are culturally and socially assigned an inferior status and are continuously demoralized by these patriarchal cultures and their phallocentric theories. Male theorists have defined females with all the negative connotations. Male writers tend to have an oppressive attitude towards females which is a great reason for their subjugation and marginalization. Moi (2003) claims, "the thinking man not only projects his desire for a reproduction of himself (for his own reflection) on to woman; he is incapable of thinking outside this specular structure" (p. 133). Within the philosophical system and structure, female sex is described as the 'other'. These female writers are speaking themselves on the behalf of those silenced women of a patriarchal society whose abilities are being suppressed under the male desires. So, the language of a woman articulates and expresses those experiences of their bodies which were devalued by the male dominant discourses. In Irigaray's opinion, the problem is that women are not allowed to speak for themselves in the patriarchal world, but the problem gets worsened when males authoritatively speak on the subject of femininity as if they have the legal right to speak for female sexuality.
Julia Kristeva (1986), a French philosopher, and critic also earned her name by challenging the man-made language structures and talks about the language differences. Kristeva believes that language is acutely dependent on the linguistics that how they define their subjects with different perceptions and connotations. Meanings are man-made. Males plot the negative meaning against females which become the major cause in the rise of power structures in patriarchal societies where ideologies represent women as passive and oppressive being. For Kristeva "the ideological and philosophical basis for modern linguistics is fundamentally authoritarian and oppressive" (as cited in Moi, 2003, p. 151). Ana I Zamorano (2005) argues that one can never make an attempt to fix the meanings as they can be changed and replaced according to the given context, therefore, for Zamorano, language is "a complex signifying process rather than monolithic system" (p. 136). Kristeva (1984) highlights the hierarchical conclusions that were forcefully connected with the language and meanings. With her attempt to break these hierarchies, she focuses on the idea of the free play of signifier where language and meanings are opened to several interpretations. Through this, she states that it is an absurd idea to define someone as a man or a woman. She writes in this connection "to believe that one 'is a woman' is almost as absurd and obscurantist as to believe that 'one is man'" (as cited in Moi, p. 163).
Afro American Feminist, Patricia Hill Collins' (2002) understanding of Afro American feminism involves black women's issues regarding gender, class race and sexuality. bell hooks (1981) , to deconstruct the conventional, mythologized negative images of the black women as aunt Jemimas, Sapphires and Jezebels that still continue to circulate in the American culture, points out that this stereotyping is the reflection of the domineering colonial outlook to keep the colonized blacks suppressed and stresses that black women must rewrite themselves through their own narratives. It is important to note though that in her seminal work Ain't I a Woman (1981), she just doesn't blame white male sexism for what she calls "imperialism of patriarchy" but also includes and refers to the black male sexist attitudes also that existed in pre-slavery era and continues through Civil Rights Movements to the present day. The efforts of women writers from marginalized nations are concentrated on challenging Western system of domination and suggesting the ways to addressing the power inequities. They suggest that both white and non white male and female must not refuse to listen to each other's stories especially when these stories tell truths that are different from the others truths.
Research Methodology
This study entirely relies upon the theoretically informed textual analysis of the selected works of Morrison (1988;1997) from historical and ecriture feminine standpoint, relying upon Belsey's suggestion (2005) that qualitative analysis always triggers new debates, (2005) seeks to offer new interpretations of the discursive reconfigurations of the cultural, historical, gendered and racial themes inscribed upon her literary and cultural artefacts, that is, women's fiction. What justifies the employment of textual analysis as the most appropriate methodological tool for this qualitative research, dealing with interpreting textual data, is that it collects data that is in the form of words and meanings and interprets it in the light of relevant critical theories and comments to enhance the understanding of the complexity of events and ideologies inscribed on the data. The two novels I have selected for the present study -Beloved and Paradise by Toni Morrison -to discuss the ways Morrison has discursively encountered the racial images and stereotypical misrepresentations of her community authentically represent the black women's emperical experiences. Since scholarly textual analysis aims to make a genuine contribution to knowledge by revealing something new, it requires on the part of the researcher a wide-ranging study of the secondary sources in order that he may draw upon variety of knowledge fields to come up with informed analysis of the complicated con(text) under discussion, accordingly this article also makes extensive use of secondary sources to support, establish, substantiate, contest and prove its knowledge claims about the complexities and interpretations of the Afro American women's literary and cultural representations. Morrison (1988), among other Afro American literary giants, stands prominent when she gives a primary importance to the tortured, enslaved and exploited blacks in her writings. She makes an unending effort to remind not only the Africans about their glorious but forgotten African values, but also the Euro-Americans who, under the delusion of their power, have been using white-governed discourse and media on the African people for centuries to enslave them and suppress their sense of dignity. For this purpose she uses literary archaeology to exhume the horrendous history of slavery as she is well-aware of the functioning of power-mechanism and the remarkable role of discourse due to her political inclination and race-consciousness. She knows the extent of difficulty in capturing the story and narrating the sufferings of those who have been deprived of their fundamental rights, and are perpetual victims of injustice and oppression of the slavery-system, still practiced by the Whites in different forms. She was fully conscious that she was going to discursively reconfigure "the unrepresented" and "the unrepresentable" as the mainstream Euro-American discourse does not allow either to tell the uncountable stories of the system of slavery or to narrate the horrendous and atrocious activities of the suppressors which are not found in numbers and statistics of slavery, but are a part of common and everyday life of the slaves. An important character in the novel is the title character Beloved. We are introduced to her body and appearance that was not even two years old when she died, "Too little to understand, too little to talk even" (Morrison, 1988, p. 5). As a young child/baby of two years old, we know her only through her body whose re-entrance in the world is a magical realism, an ecriture feminine narrative tool Morrison uses to deconstruct the hierarchical patterns of euro-centered patriarchal writing. "A fully dressed woman walked out of the water … Nobody saw her emerge or come accidently by… amid all that she was smiling" (Morrison, p. 60). Something seems abnormal and changed because of Beloved's smile. It also points out towards magical realism, an example of ecriture feminine narrative style, that asks us to suspend our reason and accept the extra-ordinary appearance as reality and go away from a phallogocentric reasoning by accepting a woman who is semi-ghost and who walks out of a river "fully dressed". Beloved's growth increases her bodily needs leading her to an immense increase in her power. At first, she is a baby that is too feeble to get out of bed but later she grows quickly, attains womanhood and seduces Paul D. She goes through a "spiritual sexual negotiation" (Henderson, 2002, p. 89) with Paul D but her innocence may not be neglected when she asks Paul D to "touch me on the inside part" and "call me my name" (Morrison,p. 137). Her innocence does not let her name any word to the act of sex. She does emphasize "her breath surgery from fingerfuls of molasses or sand-cookie crumbs" (Morrison,p. 143). She is addicted to sweets and this need to consume grows to destructive levels.
Representation of the Unrepresen Tables in Beloved
Usually, Beloved seems to be a ghost story but it is more than that as it transports the readers to so many memories where the other characters also move in their respective ways. When the identity of Beloved is disclosed to Sethe, she is "excited to giddiness by all the things she no longer had to remember" (Morrison,p. 216). When Beloved points out that the storm mixes both men and women together, she actually conveys the idea that it is the hard time that convinces the men to consider women more than a sex-doll otherwise women have always been neglected in every society. She also mentions the white men by calling them "the men without skin" whose bodies are piled on top of her, so the story of the Beloved changes from the memories of being buried or dead to the multiple narrators that lead to some other conclusions. In this regard, Henderson points out that it may be argued that Beloved's presence within the novel symbolises an immediate need that caters to both personal and communal memory (Henderson, 2002, p. 91).
This does make Beloved a uniting force for another "body" that is called a community. Seen from ecriture feminine perspective, it is obvious that these are the bodies that move the plot along but at the same time these needs of bodies as well as the speeches of bodies determine the relationships among the characters. Whether it is the relationship between Beloved and Sethe or Denver's self-discovering of a life outside the socio-religious bounds, it is the bodily hunger and speech that determined a relation. In the beginning when Beloved comes to the house, she is thirsty and drinks four glasses of water to quench her thirst as if she had crossed a desert but Denver smells another hunger in Beloved which she satisfies by giving her sweets. It is not the speech of tongue that talks about Beloved's hunger rather it is the signs of body that make Denver understand the demand of Beloved. Denver knows that the baby-ghost has come to life, so she wants to form a bond with her in order to please her. It is not that only Beloved is hungry, the hunger can also be seen in Denver who is hungry for love and affection as she is leading a lonely life.
She wants to catch and inhale sweet air from and in the mouth of Beloved in order to satisfy hunger, her hunger can be related to her mother's milk for which Beloved is also hungry. Cixous (1976) believes that breast milk is actually "white ink" which may be used by every woman writer to acknowledge the strength and power of a woman. Though some critics accuse her of using female biological essentialism yet this new language outside the pahllogocentric language helps women in raising their voice against patriarchal traditions of oppressing. Toni Morrison pays direct heed to the words of Cixous and uses breast milk and blood and water as the "ink" of her story. This 'white ink' is seen when Sethe narrates her story and beings with: "I had milk __ I was pregnant with Denver but I had milk for my body girls" (Morrison,p. 19). She narrates how the school teacher's boys made a sexual assault on her by coming closer to her to get milk from her breasts. Morrison, through the mouth piece of Sethe, uses words carefully. She does not use the words like "sexual assault", 'rape' or "beatings" rather she uses the language of body by saying that the men robbed women, especially the mothers by taking away something precious from the mothers-the thing that made them the mothers: "There was no nursing milk to call my own. I know what it is to be without the milk that belongs to you" (Morrison,p. 236).
Sethe may not consider it souring but Denver knows that the blood of her sister is mixed with her mother's milk which has caused a souring in Sethe's milk. This mixing of blood and milk is not a new tool used by any writer. In this connection, Traci C. West (1999) writes that one of the different ways slaves were tortured was a form of sexualized torture in which pregnant women and nursing mothers were whipped so brutally "that blood and milk flowed simultaneously from their breasts" (p. 278). It is quite clear that the emotions and feelings which do not find flow through tongue as the tongue has been caught by the cruel hands of slave rulers, body expresses those feelings and emotions through the running blood and flowing milk. If one part of the body (the tongue) fails to express what runs in brain, the other part comes forward as it can't be stopped. This is the very difference between body and tongue. One is bound to the external pressure and restrictions whereas the other has no limit that is why the language of body is much more powerful and affective than the tongue. Morrison expresses this very idea through Beloved that since the language of body can't be restricted so why don't the other writers, especially the female writers use this very language of body as a tool to express what is hidden in their brains. This is the effective use of this sort of innovative and expressive language that communicates the purely feminine experiences and distinguishes Morrison from others as she shuns the conventional language of phallogocentrism. She knows well that such phallogocentrism as has been conventionally used by the writers may not serve the purpose of doing justice to her stories. If she uses the conventional language of phallogocentrism, she many never be able to enjoy the space provided by Cixous' theory of ecriture feminine (1976). To break the boundaries of phallogocentrism, it is essential to break boundaries of logic and reason and to focus on bodily language. Otherwise, her story would also be drowned into the bottom of deep conventional sea.
Reclamation of Black History/Body in Paradise
Morrison's fictional world comprises of victims of slavery, racial and gender discrimination, for instance characters in Beloved (1988) and in the same way domestic and communal violence is evident in case of the group of women in Paradise (1997). The textual analysis of Paradise offers deconstructive examples galore of the Western dichotomous concepts categorizing the bodies based on the polarization of the mind and the body in which "the primary term defines itself by expelling its other" and by doing so establishes its own discursive parameters to construct an identity for itself (Grosz, 1995, p. 3). The corollary of this binaristic definitions of mind/body is the discursive construction of the black body as passive, subaltern, voiceless, non historical and irrational thus depriving the blacks of agency, subjectivity and voice of their own. Women in Paradise have different background but one thing that binds them together is that all are controlled and suppressed by the men. Cannie or Consolata is an important character in the novel who runs the convent. Her past shows that she has been taken to an Oklahoma convent by Mary Magne after being abused by phallocentric society. Morrison (1997) shows through the characters of Consolata that women, in a phallocentric society, always remain a slave of authority. This authority may also be exercised by a female. After being taken to convent, Consolata remains obedient to Mary Magna and does not do anything according to her own free-will. Her mindset is already set to follow Mary Magna's will. No doubt that Consolata is a sympathetic lady who is ready to help every needy and deserving person but she, still, is a victim of authority that runs her according to its own particular desire.
Consolata is among those women who have surrendered themselves to the others and are not going to show any resistance against aggression. She is the traditional woman who is ready to feed everyone through the milk of kindness and love. That's why, when in the second last chapter, she is attacked by men, she does not resist the man who tries to kill her. Her love for her boyfriend, (who has betrayed her) never ends till the end of her life.
Another important character is Mavis who is a negligent lady and is abused by her Patriarchal husband. Her husband Frank is alcoholic and abusive. She bears the harsh and sometimes indifferent behavior of her husband who, following the patriarchal and phallocentric ways of society, treats her violently and harshly. This thing adds to the fears of Mavis who, in order to escape from the maltreatment of her husband, tries to run-away from house. She is afraid that her husband and three children would kill her. Morrison conveys the idea through her character that fear is sleeping partner of every black-woman because the woman knows that she would never be free in the male-dominant society. Fear of punishment, fear of being left alone, fear of being killed and many more fears surround the black woman since her birth.
This fear compels her to take refuge in the Convent. Her story presages the confusion and misconceptions that arise due to the difference between visual or social perception and personal intuitive perception. Mavis is the representation of all those women who yearn to appear as competent mothers and free women but, under the callous and suppressing hands of a patriarchal society, they fail to uplift their heads and express their feelings and thoughts. Their expectations always meet a failure in a phallocentric society. But Mavis breaks this particular shell and poses to be a lady free from patriarchal bonds. This happens when she accidently kills her children. All eyes focus on Mavis and her perception is shown as muddled and unreliable as "the shine of excitement in the eyes [of people] was clear" (Morrison, 1997, p. 21). Under the conciliatory gestures of people, there is hidden a sort of hatred for Mavis. Similarly, the same shine in eyes can be seen in the photographer who comes with a reporter to make a record of her story. Despite all these facts, Morrison shows that Mavis breaks the patriarchal shell of phallocentrism. She wears sunglasses even on cloudy days which indicate the flashy life her family tries to present and her denial of reality.
Women in Ruby are not suffering from racial prejudice as much as the suppression perpetrated upon them by their own black community. Within families, the object or subject relation is dominant. Masculinity is always subjective whereas femininity is defined as object-women are object and inferior. Morrison shows that it is not the men at fault rather women themselves are the guilty ones. Power is exercised only when the weak ones refuse to resist against power. In any society, if power holds its dominant position, it is only because the people accept the reality principles determined and set by the authorities. Same is the case here in Ruby: women, since their childhood, have surrendered themselves to a phallogocentric society. They have in their minds the concept of male superiority. They have never thought in any other way. It is only women who are ready to be suppressed. They love to live in a passive condition and are not ready to revolt. In ecriture feminine tradition, Morrison wants them to speak, if not through their tongues, at least through their bodies. Women in Ruby are ignorant of their fundamental rights. They accept everything that comes from their husband or their fathers passively whether they like it or not. Mavis's life is controlled by her husband who always prevents her from having friends.
Conclusion
The articulation of the simultaneity of the multiple structures of oppression suffered by the colored women with disturbing realization that even the extrication of one form of oppression may still leave black women battling against the other equally dehumanizing form of oppression even today remains the most significant critical contribution of the black feminist thought. So, when black feminists raised their voice against the interlocking system of oppression, black men, instead of supporting their cause criticized them and placed them in the same inferioritized half of the white/black, men/women duality that the white men and women had assigned them for the perpetuation of domination. The sexual exploitation of the black women by the white men, as Toni Morrison (1988) has exemplified in case of Seth's humiliating sexual molestation at the hands of the white nephews, on the morbid white assumptions that black women are excessively lustful and temptresses has been historically used as an excuse to vindicate white men while dehumanize the black women. Black women's marathon struggle to liberate themselves from the imprisonment of sexism and racism in order to gain agency as a subject which they had been denied by the white as they were treated as objectified others of the superioritized white subjects has opened new humanist avenues of societal organization. | 2021-05-13T00:03:24.313Z | 2020-12-30T00:00:00.000 | {
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125568383 | pes2o/s2orc | v3-fos-license | Extended Second Order Sliding Mode Control for Mismatched Uncertain Systems with Only Output Measurable
Most existing Second Order Sliding Mode Control (SOSMC) approaches are achieved under assumptions that 1) all of state variables must be accessible; 2) the second derivative of all state variables must exist, even though mathematical model of systems uses the first order equations. In this paper, a new adaptive SOSMC scheme is proposed for mismatched uncertain systems in which these above assumptions are required. In this proposed method, only output variables are used in the sliding surface and controller design. The advantage of no need of all state variables in controller design makes the method more useful and realistic since it can be applied to a wider class of systems. Finally, a vertical take-off and landing aircraft at the nominal airspeed of 135 knots is simulated to demonstrate the advantages and effectiveness of the proposed approach.
Introduction
Over the past three decades, there has been an increasing research interest in Sliding Mode Control (SMC) theory and application.The main advantages of SMC are fast global convergence, simplicity of implementation, order reduction, high robustness to external disturbances and insensitivity to model errors and system parameter variations [1].Thanks to these advan-tages, the SMC theory has been successfully applied to a wide variety of practical engineering systems such as robot manipulators, aircrafts, underwater vehicles, spacecraft, flexible space structures, electrical motors, power systems, and automotive engines [1], [2] and [3].
Although the sliding mode controller guarantees robustness with respect to uncertainties and external disturbances, chattering is its main drawback.Chattering is the high frequency finite amplitude oscillations occurring because of the discontinuous control signal used in the SMC [4].Such chattering has many negative effects in practical applications since it may damage the control actuator and excite the undesirable unmodeled dynamics, which probably leads to unforeseen instability [4].Many authors have applied various techniques to reduce chattering problem across the sliding surface.Recently, some good results have been published in high quality journal such as [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18] and [21].The authors of [5] have presented a direct way to reduce chattering problems by inserting a fixed or variable boundary layer near the sliding variable so that a smooth continuous control replaces the discontinuous one when the system is inside the boundary layer.This approach can produce a chattering-free system but a larger boundary layer width results in larger errors in control accuracy and a finite steady-state error may occur.Another approach to eliminate the chattering is carried out by using fuzzy control with sliding mode controller [6], using low-pass filtering [7] or nonlinear reaching law [8].This method can give a chatteringfree system but a finite steady-state error may remain.
One of the most effective methods to avoid chattering problems is to use the Second-Order Sliding Mode Control (SOSMC).The basic idea of the second order sliding mode controller is that the discontinuous sign function is made to act on the time derivative of the control inputs and the actual control signal obtained after integration is continuous and hence chattering is removed [9].In addition, SOSMC allows driving to zero the sliding variable and its consecutive derivatives in the presence of the disturbances/uncertainties increasing the accuracy of the sliding variable stabilization [10].Thanks to these advantages, the SOSMC with finite-time convergence has been successfully implemented for solution of real problems [11] and [12].Another approach given in [13] was to present a modified second-order sliding mode control for single-input nonlinear systems.This study guarantees the finite time reaching of the sliding manifold and chattering reduction.The SOSMC proposed in [13] was extended by [14] for a class of uncertain multi input nonlinear systems but the disturbances were not considered in the above approach.
In [15], the second-order sliding mode control approach with additional capabilities of learning and control adaptation was developed to estimate and compensate for the uncertainty affecting the system's dynamics.This technique is capable of reducing the discontinuous control effort to an arbitrarily small quantity.However, the approach given in [15] could not be applied for systems with unknown upper bounds of uncertainties.The study of [16] proposed a robust adaptive SOSMC scheme for a class of uncertain nonlinear systems where the upper bounds of uncertainties are not required to be known in advance.As a result, a finite-time convergent second-order sliding mode is established and the chattering problem is eliminated.In [17], an adaptive second order sliding mode control law is proposed for the control of an electro pneumatic actuator.In order to reduce the overshoot and the settling time, the adaptive second order sliding mode controller with a nonlinear sliding surface is presented in [18].The authors of [19] have proposed a chattering free adaptive sliding mode controller for stabilizing a class of multi-input multi-output systems.This approach can ensure asymptotical stability of the overall system and eliminate chattering in the control input.In [20], based on the linear quadratic regulator method, an optimal second order sliding mode controller was proposed for a class of matched uncertain systems.By designing a new sliding surface, the approach given in [21] can solve both the chattering and singularity problems in sliding mode control.A second-order sliding mode control method to handle sliding mode dynamics with mismatched term was presented in [22].In [23], a robust chattering-free control scheme was proposed using second-order fast terminal sliding mode control technique for the tracking problem of a class of uncertain systems with matched and mismatched uncertainties.
However, it is worth pointing out that most of the previous results have been developed under the assumption that all the system states are available for the control law.It may be impossible or prohibitively expensive to measure all of the process variables in some practical systems [24].For example, it is difficult to measure the variables describing the flexible motion, the modal position, and the velocity of flexible spacecraft [25].Thus, it is very important to establish a new adaptive SOSMC method to control mismatched uncertain systems via output feedback.Herein, we intent to use the output information completely in the sliding surface and controller design but still remain the advantages of SOSMC such as the chattering-free, the maximum convergence time interval, and the dimension of neighbourhood of the origin to which the controlled trajectory converges [14].
In this paper, we extend the concept of second order sliding mode controller, introduced by [19], [22], [23] and [27], for the aim of stabilizing mismatched uncertain systems where only output variables are accessible.The main contributions of this paper are as follows: • A new Lemma and a novel adaptive law are established for the aim of controller design using only output variables.
• New sufficient conditions in terms of Linear Matrix Inequalities (LMI) are derived such that the equivalent reduced-order system in the sliding mode is asymptotically stable.
• The two major assumptions by [19], [22], [23] and [27] (that all of state variables must be accessible, and that the second derivative of all state variables must exist) are both eliminated.Therefore, the proposed method can be applied to a wider class of mismatched uncertain systems.
System Description and Preliminary Results
Consider the following mismatched uncertain systems: Here x ∈ R n , u ∈ R m and y ∈ R p denote the state variables, inputs and outputs, respectively.A ∈ R n×n is state matrix, B ∈ R n×m is the input matrix and C ∈ R p×n is the output matrix.The terms ∆A and ξ(x, t) represent the system matrix and the input matrix uncertainties, respectively.We assume that: Assumption 1.The matrix pair (A, B) is completely controllable.
The mismatched uncertainty ∆A(x, t) is a norm-bounded time varying uncertainty as follows: where D and E are known constant real matrices with appropriate dimensions that characterize the structure of the uncertainty, and F(x, t) is a norm-bounded unknown matrix.Assumption 3. rank(CB) = m.
From [28], Assumption 3 implies that there exists a non-singular linear coordinate transformation z = T x such that the triple (A, B, C) with respect to the new coordinates has the structure where Assumption 4 implies that there exist matrix K such that the matrix A 1 = Ã1 − Ã2 KΞ is stable.From [28], the coordinate transformation z = T x where will transform the triple (A, B, C) to the following form in the new coordinate system z where Remark 1: In [19], [22], [23] and [27], all of state variables x ∈ R n must be accessible and the second derivative of all state variables ẍ exist, even though mathematical model of systems is of the first order.The proposed method needs only a subset of state variables y ∈ R p to be accessible and the second derivative of output variables ÿ exists.Therefore, the proposed approach can be applied to a wider class of mismatched uncertain systems.
Remark 2: The output feedback SOSMC scheme is proposed in [26].However, there are three major conditions set by [26]: • The system under consideration is assumed to be matched.
• The exogenous disturbances are bounded by a known constant value.That is f ≤ π where π is known.This condition is quite restrictive.
• The sliding matrix F satisfies that the matrix FCAB is invertible to guarantee sliding condition S(t) = F y(t) + w(t) = 0.This limitation is really strong.
Remark 3: The SOSMCs using output variables were subject of many recently researches [29] and [30].However, all these methods require more hardware and increase system dimension.In this paper, an adaptive output feedback SOSMC scheme is proposed for mismatched uncertain systems where above limitations are eliminated.
Adaptive Output Feedback Second Order Sliding Mode Control Design
In this section, we introduce a systematic design procedure of an adaptive output feedback Second Order Sliding Mode Control (SOSMC) scheme.There are three steps involved in the design of an adaptive output feedback SOSMC for system, see Eq. (1).In the first step, a sliding surface is designed to depend on only output variables.In the second step, we derive appropriate Linear Matrix Inequalities (LMI) stability conditions by the Lyapunov method to guarantee that the system in the sliding mode is asymptotically stable.In the third step, we design an adaptive output feedback second order sliding mode controller in a way such that the system states reach the sliding manifold in finite time and remain it thereafter.
Sliding Surface Design
From Eq. ( 11), Eq. ( 12) and Eq. ( 13) of paper [28], it follows that under Assumptions 3 and 4, there exists a non-singular matrix T such that in the new coordinates z = T x the system Eq.(1) can be described as: and where It follows from Eq. ( 5) that and For the systems Eq. ( 5) and Eq. ( 6), consider a sliding surface σ(y(t)) where in which P ∈ R (n−m)×(n−m) is defined later and the matrix Ψ ∈ R m×(n−m) is selected such that the matrix K 2 ∈ R m×m is non-singular.According to Eq. ( 6), the sliding surface Eq. ( 9) can be rewritten as: where N = O (p−m)×(n−p) I (p−m)×(p−m) .From Eq. ( 11) and since K 2 ∈ R m×m is non-singular, in the coordinate z, the sliding surface Eq. ( 9) can be described by: Using Eq. ( 12), the dynamic equation in sliding mode is:
Stability Analysis of Sliding Motion
In last section, we have designed an output sliding surface.There are still two important tasks that should be done.The first task is to derive appropriate LMI stability conditions by the Lyapunov method to guarantee that the sliding mode dynamics Eq. ( 13) is asymptotically stable.The second task is to design an adaptive output feedback SOSMC in a way such that the system states reach the sliding manifold in finite time and stay on it thereafter.Now, we are going to do the former task by considering the following LMI: where G ∈ R (n−m)×(n−m) is general and non-zero matrix, P ∈ R (n−m)×(n−m) is any positive matrix, Θ = ϕG T D 1 D T 1 G and the scalar ϕ > 0.Then, we can establish the following theorem.
Proof : For the sliding mode dynamics Eq. ( 13), consider a candidate Lyapunov function function where P > 0 satisfies Eq. ( 14).The time derivative of V along the trajectories of Eq. ( 13) is given by From Eq. ( 16), if (A 1 +D 1 F E 1 ) T P + P (A 1 +D 1 F E 1 )<0 then V < 0 and the sliding mode dynamics Eq. ( 13) is asymptotically stable.
Before proving V < 0 , we recall the following Lemmas.
Lemma 1. [31]: Let X, Y and F be matrices of compatible dimension then XF Y + Y T F T X T < ϕ −1 XX T + ϕY T Y for any F satisfying F ≤ 1 and a scalar ϕ > 0.
Lemma 2. [31]: Given a symmetric matrix W and two matrices Γ and, Σ and consider the problem of finding some matrix G such that W + ΣGΓ + (ΣGΓ) T < 0.
Denote Σ ⊥ and Γ ⊥ any matrices whose columns form the bases of the null spaces of Σ and Γ, respectively.Then the above inequality is solvable for G if and only if Σ ⊥ W Σ ⊥T < 0, Γ T ⊥ W Γ T ⊥T < 0. Now, we are going to prove V < 0. Let us first define is defined in LMI Eq. ( 14).Then, we have Applying Lem. 1 to Eq. ( 17), we achieve where Θ = ϕG T D 1 D T 1 G and the scalar ϕ > 0. Using Schur complement formula, LMI Eq. ( 14) can be rewritten as From Eq. ( 18) and Eq. ( 19), it can be observed that It follows from Eq. ( 20) and Lem. 2 that Since the fact W = 0 P P 0 , and T , we obtain: According to Eq. ( 16) and Eq. ( 22), it is obvious that Note that Eq. ( 23) verifies that Eq. ( 14) holds, which further implies that sliding motion is asymptotically stable.The following new Lemma is derived for controller design using only output variables.
Lemma 3. Consider the reduced-order system Eq.( 7).If the matrix A 1 is stable then z 1 (t) is bounded by η(t) for all time, where η(t) is the solution of where k D 1 E 1 +λ < 0, the scalar k > 0 and λ is the maximum eigenvalue of the matrix A 1 .
Proof : Solving Eq. ( 7) gives The stable matrix A 1 satisfy the constraint where k > 0 and λ < 0 are defined in Eq. ( 3).According to Eq. ( 25) and Eq. ( 26) we have Multiplying both sides of Eq. ( 27) by exp(−λt), gives Letting h(t) is the right term of Eq. ( 28) and taking the time derivative of h(t), yields According to Eq. ( 28) and Eq. ( 29) we achieve Multiplying both sides of Eq. ( 30) by exp(−k Integrating both sides of Eq. ( 31), we obtain where where η(t) is defined in Eq. ( 3).Therefore, it is easy to conclude that η(t) ≥ z 1 (t) for all time, if η(0) sufficiently large.
Adaptive Output Feedback Second Order Sliding Mode Controller Design
In the last section, we dealt with the first and second elements of the design process.In this section, we design an adaptive output feedback second order sliding mode controller such that the system states reach the sliding manifold in finite time and stay on it thereafter.Let us begin with defining the sliding manifold s(t) such that where X ∈ diag(χ 1 , χ 2 , ...χ m ) is any diagonal matrix.
The main idea behind the second-order sliding mode is to act on the second-order derivative of the sliding variable σ rather than the first derivative as in conventional sliding mode.The second-order sliding mode is determined from the basic equality condition σ = σ = 0 reaches in finite time, whereas the proposed controller reaches the condition asymptotically.
According to Eq. ( 9), we obtain Since KC −1 2 y = K 2 z 2 and Eq. ( 8), Eq. (37) can be rewritten as: where Assumption 5.The disturbance ξ(t) of Eq. ( 38) in the domain of interest satisfies where a i are unknown positive constants, r is a designed positive integer.
The proposed adaptive output feedback second order sliding mode controller for tackling the system uncertainty is designed as follows: where the scalar α > 0, , and H 1 H 2 = T −1 .The adaptive gains âi (t) are given by ȧi The time function η(t) is the solution of Eq. ( 3) and q i , qi are positive constants.The major drawback of sliding mode control is so-called chattering phenomenon.Such a phenomenon consists of the oscillation of the control signal, tied to the discontinuous nature of the control strategy, at a frequency and with an amplitude capable of disrupting, damaging or, at least, wearing the controlled physical system.It should be pointed out that the controller Eq. ( 40) is a continuous control input and uses only output variables.Hence the undesired high frequency chattering of the control signal is eliminated.This is a new contribution of the proposed method.Now let us discuss the reaching conditions in the following theorem.
Theorem 2. Consider the uncertain dynamic system defined by Eq. (1) with the assumptions 1-4.If the sliding manifold and the adaptive sliding mode controller are designed as Eq.(34) and Eq. ( 40), respectively.Then, the system states reach the sliding manifold s(t) in finite time and stay on it thereafter.
Proof : Define a Lyapunov function candidate as follows: where ãi (t) = a i −ã i (t), i = 0, 1, ..., r are the estimation errors of the adaptive gains.Now taking the derivative of V (t) yields Using Eq. ( 7), Eq. (38) and Eq. ( 43) and property AB ≤ A B , it generates From Assumption 4 and x ≤ H 1 z 1 + H 2 z 2 where H 1 H 2 = T −1 .We can obtain that c 2018 ADVANCES IN ELECTRICAL AND ELECTRONIC ENGINEERING Equation (11) implies that According to Lem. 3, we have From Eq. (41), Eq. (45), Eq. ( 46) and Eq. ( 47), it can be observed that Substituting the controller Eq. (40) into Eq.( 48), we achieve Then, from Eq. ( 49), it is easy to see that the uniform ultimate boundedness can be guaranteed.
The proposed adaptive SOSMC, using the output information completely in the sliding surface and controller design, offers following advantages.Firstly, conservatism is reduced and the robustness is enhanced.Secondly, an improved transient performance can be obtained without the knowledge about the upper bound of the system uncertainties.Finally, the chattering in the control input is removed.
Design procedure: The proposed adaptive output feedback SOSMC scheme can be simultaneously designed by the following steps.
Numerical Example
In order to demonstrate the validity and effectiveness of the proposed method, in this section, we are going to apply the adaptive output feedback SOSMC given in previous sections for a Vertical Take-Off and Landing (VTOL) aircraft at the nominal airspeed of 135 knots, which is modified from [19]. where is the horizontal velocity (knots), x 2 is the vertical velocity (knots), x 3 is the pitch rate (degrees per second) and x 4 is the pitch angle (degrees), u 1 is the collective pitch control, u 2 is the longitudinal cyclic pitch control.It is assumed that x 1 , x 2 , and x 4 are the output signals.
The mismatched uncertainty is given as ∆A = DF E with D = 1 1 1 0 T , E = 0 1 1 1 and The disturbance is assumed to satisfy the following condition ψ(t) ≤ 2 + 0.1 x .For this work, the following parameters are selected as follows: α = 300.1,ϕ = 10.0109, and k = 1.001.The controller for the system Eq.(50) and Eq. ( 51) is the solution of the following equation: The initial conditions for the above system are selected to be x(0) = 2 −2 1 1 T and u(0) = 0 0 T .The system states and the control input of the VTOL aircraft system using the proposed adaptive output feedback SOSMC are shown in Fig. 1, Fig. 2 and Fig. 3.It is evident from Fig. 1 that the proposed adaptive output feedback SOSMC produces faster convergence of the system states to equilibrium as compared to the method proposed by [19].It is observed from Fig. 2 and Fig. 3 that the actual control input obtained by the proposed method is smooth and chattering free.Convergence of the sliding surface is shown in Fig. 4 and Fig. 5. Figure 4 and Fig. 5, clearly show that the proposed sliding surface is smooth and approach to equilibrium point quickly.
Remark 4: The method proposed by [19] cannot be applied for the system Eq.(50) and Eq.(51) if the state variable x 3 is unmeasurable.This limitation has been removed by the proposed adaptive output feedback SOSMC Eq. ( 53) because the proposed controller Eq. (53) only uses three output variables (x 1 , x 2 , and x 4 ).
Remark 5: The mismatched parameter uncertainties in the state matrix of the system Eq.(50) and Eq. ( 51) are non-linear and time-varying.Thus, the approaches given in [26] could not be applied for the system defined by Eq. (50) and Eq.(51).
Conclusion
This paper has presented a new adaptive Second Order Sliding Mode Control (SOSMC) for mismatched uncertain systems where only output information is available.The proposed SOSMC is guaranteed that the system in sliding mode is asymptotically stable and the state trajectories reach the sliding manifold in finite time and stay on it thereafter.Furthermore, system performance using the proposed control is good without and no chattering phenomenon exists.The most significant advantage of the proposed SOSMC scheme is that the measurement of all the system state variables which are required in most existing SOSMCs is removed.This is valuable for cases in which the system state variables are unavailable.Uncertain
Assumptions 2 and 3
can be shown to hold.The coordinate transformation z = T x is given by: | 2019-04-22T13:13:15.565Z | 2018-05-12T00:00:00.000 | {
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4086255 | pes2o/s2orc | v3-fos-license | Erratum to: JAK3-STAT pathway blocking benefits in experimental lupus nephritis
Unfortunately, after publication of this article [1], it was noticed that the name of Juliana Draibe was listed incorrectly. The corrected name can be seen in the author list above and the original article has been updated to reflect this change.
Background
Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disorder characterized by loss of tolerance against nuclear autoantigens, anti-double-stranded DNA (anti-dsDNA) antibody production, immune complex (IC) deposition, and leukocyte infiltration in many target organs, as well as activated B and T cells [1,2]. Lupus nephritis (LN) is an IC glomerulonephritis categorized as one of the most serious complications of SLE and is also one of the strongest predictors of a poor prognosis. LN can lead to severe proteinuria, hypertension, chronic renal failure, and, finally, end-stage kidney disease.
It is well known that the cytokine milieu is integrally involved in the pathogenesis of autoimmune diseases [3,4]. Multiple cytokines, such as interferons (IFNs), tumor necrosis factor (TNF)-α interleukin (IL)-6, IL-12, IL-10, and IL-17, are implicated in the initiation, progression, and development of LN. Serum levels of several of them have been found to be elevated in patients with active SLE and in murine LN models [5][6][7][8], and abnormally high levels are associated with more severe disease and severity [9,10]. IL-6 is a proinflammatory cytokine that plays a central regulatory role in the development, maturation, survival, and immunoglobulin secretion of long-lived plasma cells [11]. IL-17 amplifies the immune response by inducing the local production of chemokines and cytokines, recruiting neutrophils and monocytes, augmenting the production of autoantibodies, and aggravating the inflammation and damage of target organs [12].
One of the best-understood mechanisms by which cytokines activate signals, thus eliciting specific responses in target cells, involves enzymes such as Janus kinases (JAKs) and transcriptional factors such as signal transducers and activators of transcription (STATs). The JAK-STAT signaling pathways are used by all type I and type II cytokine receptors. Following ligation, JAK phosphorylates the cytoplasmic tail of its receptor, which becomes activated, leading to the recruitment of STATs. Afterward, STATs translocate into the nucleus, where they regulate the expression of numerous genes [13]. The pathological consequences of deregulated JAK/STAT signaling have been widely documented in many animal models of chronic inflammatory and autoimmune diseases as well as cancers [14]. Some polymorphisms of JAK/STAT genes have also been associated with susceptibility to SLE [15,16]. This association with severe immune dysfunction reveals the key role of this pathway in the induction and regulation of immune responses.
The JAK3 inhibitor CP-690,550 is a drug currently approved for the treatment of rheumatoid arthritis. It is currently also being evaluated for the treatment of other autoimmune diseases, as well as for the prevention of organ transplant rejection [17,18]. Very few studies have assessed this family of drugs in the context of LN. CP-690,550 is an orally available compound that specifically binds to the adenosine triphosphate (ATP)-binding pocket of JAK3, though recent studies have shown that CP-690,550 is also able to bind to the ATP pocket in both JAK1 and JAK2 [19,20]. Therefore, CP-690,550 is currently categorized as a pan-JAK inhibitor preferentially inhibiting JAK3 and JAK1, and to a lesser extent JAK2.
In contrast to the relatively ubiquitous expression of JAK1, JAK2, and Tyk2, JAK3 has a more restricted and regulated expression. JAK3 is selectively expressed in hematopoietic cells, natural killer cells, thymocytes, T and B cells, and myeloid cells, and mutation in this kinase results in a loss of immunological function and in severe combined immunodeficiency [13]. We hypothesized that blocking the enzymatic activity of JAK3 might also result in immunosuppression, thereby offering a novel, attractive, and promising strategy in the treatment of inflammatory immune-mediated diseases. Therefore, in the present study, we examined the effects of blocking the JAK-STAT pathway on disease progression and renal lesions in NZB/WF1 mice via in vivo administration of CP-690,550. We further evaluated whether this blockade interferes only locally in the progression of LN or also in downstream mechanisms of SLE development.
Mice, study design, and follow-up
Five-month-old NZB/WF1 mice (JAX®; Charles River Laboratories, Barcelona, Spain) were randomly assigned to four groups. At 6 months of age, treatment was initiated as follows, by group: intraperitoneal cyclophosphamide (CYP) 50 mg/kg/10 days (n = 14), subcutaneous CP-690,550 (CP, XELJANZ® [tofacitinib]; Pfizer, New York, NY, USA) 48 mg/kg/day (n = 8), oral mycophenolate mofetil (MMF, CellCept®; ROCHE FARMA, Madrid, Spain) 30 mg/kg/daily (n = 8), and subcutaneous PBS treatment (n = 14) as a control group. Mice were treated for 12 weeks. Their body weight was determined twice monthly from the beginning to the end of follow-up. Mice were placed in metabolic cages to collect 24-h urine specimens before the onset of treatment, and monthly thereafter. Blood was obtained from the tail vein at monthly intervals. Kidneys were processed for histological and biochemical studies at the end of the study or at death.
The experiments were carried out in accordance with current European Union legislation on animal experimentation and were approved by the animal experimentation ethics committee, the University of Barcelona Institutional Ethics Committee for Animal Research, and the Animal Experimentation Commission of the Generalitat de Catalunya (Catalonian government). The mice were housed in a room at constant temperature with a 12-h dark/12-h light cycle, were given free access to water, and were fed a standard laboratory diet.
Proteinuria, albuminuria, and renal function
Twenty-four-hour urinary protein was determined by Pyrogallol red reaction (AU400 Clinical Chemistry System; Beckman Coulter, Brea, CA, USA) in the Veterinary Clinical Biochemistry Laboratory of Universitat Autonoma de Barcelona. Twenty-four-hour urinary albumin was determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's instructions.
RNA extraction, reverse transcription, and gene expression analysis using quantitative real-time polymerase chain reactions For molecular studies, the kidney was immediately snapfrozen in liquid nitrogen and stored at −80°C. RNA was extracted from kidneys using the PureLink™ RNA Mini Kit (Ambion/Thermo Fisher Scientific, Barcelona, Spain) according to the manufacturer's instructions. RNA purity was analyzed using a NanoDrop ND-1000 V3.3 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA was stored at −80°C. A total of 500 ng of RNA was used for reverse transcription with a highcapacity cDNA reverse transcription kit (Applied Biosystems, Warrington, UK) in accordance with the manufacturer's instructions. Tissue expression levels of the following mediators of immunity and inflammation were quantified by TaqMan real-time polymerase chain reaction (ABI Prism® 7700, Applied Biosystems, Spain) using the comparative C t method: complement component 3 (C3), chemokine (C-C motif ) ligand 2 (CCL2), CCL5, CD40L, IL-2, IL-6, Toll-like receptor 9 (TLR9), vascular cell adhesion molecule (VCAM)-1, STAT1, STAT2, STAT3, STAT4, and STAT5a.
Plasma ELISA for anti-DNA antibodies and serum cytokine analysis Levels of anti-DNA antibodies were measured using a commercially available ELISA kit (Alpha Diagnostic International, San Antonio, TX, USA) according to the manufacturer's instructions. Serum IL-12, TNF-α, IFN-γ, and monocyte chemoattractant protein (MCP)-1 cytokines were measured using the BD FACSCanto flow cytometer with a cytometric bead array kit (BD CBA mouse inflammation kit) according to the manufacturer's instructions (BD Biosciences, San Jose, CA, USA). Data were acquired and analyzed using BD FCAP software and CBA software (BD Biosciences). Serum IL-17 (R&D Systems, Minneapolis, MN, USA) and IFN-α (PBL Assay Science, Piscataway, NJ, USA) cytokine levels were quantified using commercially available ELISA kits according to the manufacturers' instructions.
Renal lupus histopathology
For histological analyses, 1-to 2-mm-thick coronal slices of kidney tissue were fixed in 4 % formaldehyde and embedded in paraffin. For light microscopy, 3-to 4-μmthick tissue sections were stained with hematoxylin and eosin stain and periodic acid-Schiff stain. To determine the extent of renal damage, two blinded pathologists analyzed all kidney biopsies. Typical glomerular active lesions of LN were evaluated: mesangial expansion, endocapillary proliferation, glomerular deposits, extracapillary proliferation, and interstitial infiltrates, as well as tubulointerstitial chronic lesions, tubular atrophy, and interstitial fibrosis. Lesions were graded semiquantitatively using a scoring system from 0 to 3 (0 = no changes, 1 = mild, 2 = moderate, and 3 = severe). Finally, a total histological score was derived from the sum of all the described items.
Paraffin-embedded tissue sections were also stained for CD3 (Abcam, Cambridge, UK). The sections were blocked and labeled with immunoperoxidase using a VECTASTAIN ABC kit and an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's protocol. Peroxidase-conjugated antibody staining was followed by diaminobenzidine substrate development (Sigma-Aldrich, Madrid, Spain).
Renal immunofluorescence studies
Slices of kidney were fixed in 4 % paraformaldehyde, embedded in Tissue-Tek® O.C.T. compound (Sakura Finetek, Alphen aan Den Rijn, the Netherlands), and stored at −80°C. Fluorescent staining of 5-μm cryostat sections was used for confocal microscopy to quantify glomerular immunoglobulin G (IgG) and C3 deposition. Sections were directly stained with fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG (Sigma-Aldrich) and FITC-conjugated C3 (Nordic-MUbio, Susteren, the Netherlands). For analysis of C3 and IgG deposition, at least ten glomeruli were visualized and photographed with an immunofluorescence confocal microscope (Leica TCS SL spectral confocal microscope; Leica Microsystems, Mannheim, Germany). Fluorescence was quantified and normalized with Simulator-Leica confocal software (Leica Microsystems) and expressed as mean fluorescence intensity.
To quantify kidney macrophages, F4/80 immunohistochemistry was used. Briefly, 3-μm-thick kidney tissue sections embedded in paraffin were incubated with primary antibody antimouse F4/80 (eBioscience, San Diego, CA, USA) and incubated overnight at 4°C. Staining was visualized using a secondary Alexa Fluor 546 dye (Molecular Probes/Thermo Fisher Scientific, Eugene, OR, USA). Nuclei were stained blue with DRAQ5 (eBioscience). To determine macrophage infiltration, at least 15 high-power fields were counted, and the number of positive cells was determined and expressed as a mean value.
Statistical analysis
Overall survival was analyzed with the Kaplan-Meier method. One-way analysis of variance with post hoc tests were performed to compare proteinuria and anti-dsDNA antibodies throughout the follow-up, and gene expression and circulating cytokines were evaluated at the time the mice were killed. To compare histological data, a nonparametric Kruskal-Wallis test was used. A p value <0.05 was considered significant. Data are expressed as mean ± SEM.
JAK3 inhibition prolongs survival and ameliorates renal function
To examine the effects of a JAK3 inhibitor on advanced LN in mice, we treated 6-month-old NZB/WF1 female mice with overt renal disease with CP-690,550 and compared this treatment with MMF and CYP as standard therapies. Cumulative survival analyzed with the Kaplan-Meier method was 100 % for the CYP and MMF groups, 85 % for the CP-690,550 group, and 76 % for the control group at the end of the follow-up.
NZB/WF1 mice at 5 months old had slight to moderate proteinuria and albuminuria, which indicates LN. As the mice became older, without treatment they typically had severe disease, as evidenced by a progressive increase in proteinuria and albuminuria levels (Fig. 1). Twelve weeks of CP-690,550, MMF, and CYP administration resulted in significant reduction of urinary protein and albumin excretion compared with control animals. At week 36, those parameters were significantly decreased in all treated animals. Additionally, the results showed that CP-690,550-treated mice had levels of proteinuria and albuminuria similar to those of mice that received standard therapies.
JAK3 inhibition affects the production of anti-dsDNA autoantibodies
Serum levels of anti-dsDNA autoantibodies were determined at several time points after the beginning of treatment. As shown in Fig. 2, by age 28 weeks, serum titers of anti-dsDNA autoantibodies progressively increased in All treatment regimens appeared to have some inhibitory effect on autoantibody production. There was a trend toward anti-dsDNA antibody reduction after week 32. Anti-dsDNA titers in the CYP-, MMF-, and CP-690,550-treated animals were significantly less than those in control mice at week 36 (p < 0.05). Note that while CYP broke the production of anti-dsDNA antibodies from the beginning of treatment, CP-690,550 treatment had a more profound effect and was more effective at the end of the treatment.
JAK3 inhibition prevents glomerular and tubular lesions
Renal histological analysis was performed on all surviving animals at the end of the study (Fig. 3). Typical LN lesions were seen in control mice that developed significant glomerulonephritis and interstitial inflammation. Regarding treatments, the CYP group showed a reduction in all evaluated lesions compared with vehicletreated mice, exhibiting a pronounced reduction in endocapillary proliferation, glomerular deposits, and tubular atrophy. In the MMF group, glomerular deposits, extracapillary proliferation, and interstitial fibrosis were absent. Nevertheless, MMF animals showed important mesangial expansion and interstitial infiltrate compared with vehicle animals. CP-690,550 treatment induced a drastic reduction in endocapillary proliferation, glomerular deposits, and interstitial infiltrate, while other lesions, such as extracapillary proliferation, tubular atrophy, and interstitial fibrosis, were completely absent. Mean histological scores were as follows: CYP = 2.6 ± 0.8, MMF = 1.6 ± 0.5, and CP = 2.6 ± 1.2. These scores were statistically different with respect to the control group (control = 8.6 ± 1.1; p < 0.001).
Complement and IgG glomerular deposits are reduced by JAK3 inhibition
Intrarenal deposits of IgG and complement in the glomeruli were significantly diminished in all treated animals compared with the control group (Fig. 4). IgG glomerular deposits were lower in the CP group than in the control and CYP groups. C3 glomerular deposits were also reduced in all treatment groups, although in this case CYP treatment was more effective than MMP and CP-690,550 treatment.
Macrophages and T-cell infiltration in the glomeruli are hampered by JAK3 inhibition
To better characterize the inflammatory cell infiltration observed with conventional histology, F4/80 (macrophage) and CD3 (T-cell) surface markers were analyzed (Fig. 5). The number of cells that stained positive for F4/ 80 was significantly reduced in all treated groups compared with the control group. As can be seen in the photomicrographs in Fig. 5, macrophages were localized mainly around the glomeruli close to the Bowman's capsules and in the interstitial area.
T-lymphocyte infiltration evidenced by CD3 immunostaining showed that, again, all treatments affected kidney CD3 infiltration. Infiltrating CD3 + cells were localized in the tubule-interstitium space, where dendritic cells and other inflammatory cells are commonly found.
JAK3 inhibition modulates renal inflammatory gene expression
Expression analysis of different inflammatory genes in the kidney (Table 1) revealed that there was a significant upregulation in their expression compared with healthy animals. The JAK-STAT pathway is key in the pathogenesis of LN, so vehicle-treated control animals displayed an elevated expression of STAT genes that signal downstream in the cascade of JAK receptors (STAT1, STAT2, STAT3, STAT4, and STAT5a). All treatments induced a reduction in expression of these genes, achieving almost healthy levels, which indicates good inhibition of the pathway by all the compounds, especially CP-690,550. C3 gene expression, as a manifestation of local complement synthesis, was significantly reduced in all treatments. These results paralleled the C3 glomerular deposits. CCL2 and CCL5 are genes related to the recruitment of several inflammatory cells into inflammatory sites, and they play an important role in the recruitment of monocytes, T cells, dendritic cells, eosinophils, and so forth. The results showed that all of these genes were diminished in treated animals. Another gene that participates in cell adhesion is VCAM. Its expression was reduced by all treatments, according to the kidney cell infiltration results. The costimulatory pathway was also affected by CYP, MMF, and CP-690,550 An intense local effect in inflammatory cytokine expression was observed. IL-2 and IL-6 were significantly reduced in all treatment groups. There was no evidence of activation of TLR4 (data not shown). TLR9, a sensor of dsDNA, was overexpressed in vehicle-treated animals and was strongly reduced by all treatments.
JAK3 inhibition decreases proinflammatory systemic circulating cytokines
To determine whether the different treatments modified the systemic inflammatory response, we measured serum levels of some proinflammatory cytokines (Fig. 6). LN induced an increase in the secretion of different pathogenic proinflammatory cytokines, such as IL-12, IL-17, TNF-α, MCP-1, IFN-α, and IFN-γ, compared with healthy animals (data not shown). CYP treatment induced a reduction in almost all these cytokines. Of note, CYP treatment did not affect IFN-α circulating levels. MMF treatment appeared to be less immunomodulatory, considering secretion of this cytokine. JAK3 inhibition showed a trend toward reducing all the evaluated Th1 cytokines, but it is noteworthy that there were significant reductions in the secretion of TNF-α, IFN-α, and IL-17.
Discussion
With recent insight into the pathological role of JAK-STAT proteins in the inflammatory process, it has Fig. 4 Immunofluorescence analysis of renal immunoglobulin G (IgG) and complement component 3 (C3) deposits. Deposits of renal C3 (a) and IgG (b) were quantified with confocal microscopy, and the results were expressed as mean fluorescence intensity (MFI). All treatments reduced glomerular deposits. c Representative photomicrographs of C3 deposits (×630 original magnification) for each group. d Representative photomicrographs of IgG deposits (×630 original magnification) for each group. Data are expressed as mean ± SEM. *p < 0.05 vs control. CP CP-690,550; CYP cyclophosphamide; MMF mycophenolate mofetil become feasible to target this pathway in order to modulate their pathogenic effects. In this study, we have demonstrated that treatment of NZB/WF1 mice, a wellrecognized experimental murine model of autoimmune nephritis, with the JAK3 inhibitor CP-690,550 resulted in improved animal survival, delayed the progression of the disease, and ameliorated renal function, preventing kidney cell infiltration and complement deposition. We have also proven that treatment with CP-690,550 reduced the severity of typical tubular and glomerular lesions and that JAK-STAT inhibition had an impact on autoantibody formation, as illustrated by a reduction in circulating anti-dsDNA antibody titers, a hallmark of the disease. As expected, either renal mRNA expression or circulating inflammatory cytokine signaling through the JAK-STAT pathway was diminished.
CYP has been used as a standard treatment for SLE and LN, but its toxicity and relatively low effectiveness in preventing disease relapse have made its use less common; more than 30 % of patients with LN relapse within 3 years after cessation of therapy. In addition to CYP, we included MMF in our experimental design, which nowadays is becoming a reference drug for the treatment of patients with LN [21]. In our present study, we have proven in an experimental mouse model that both drugs exert similar potent effects in all the evaluated parameters, as previously reported in patients [21].
The role played by proinflammatory cytokines in the pathogenesis of LN has been clearly established [22,23]. One of the promising developments in the last decade has been the identification of the function of the IL-23/Th17 pathway in autoimmune disease initiation, progression, [24]. A strong correlation was observed in serum levels of IL-17 in patients with SLE, suggesting that IL-17 may drive activation of diverse autoimmune pathways. Many of the cytokines involved in LN and other autoimmune diseases, including IL-17, signal through receptors associated with JAKs, and CP-690,550 targets JAK3, which plays a pivotal role in the beginning of the inflammatory cytokine signaling pathway. In this sense, the present study demonstrates that inhibition of the JAK-STAT pathway is essential in preventing proinflammatory cytokines from fulfilling their role.
LN is characterized by the presence of circulating autoantibodies as well as deposition of immune complexes or components in renal tissue [25,26]. As our results show, CP-690,550 treatment significantly reduced the deposition of IgG and C3 in the glomeruli, probably as a result of the inhibition of circulating autoantibodies, as reflected by the reduced anti-dsDNA serum levels in treated animals. Also, our results suggest that CP-690,550 is more effective than CYP or MMF in the inhibition of anti-dsDNA autoantibody formation. In other studies, researchers have demonstrated the influence of CP-690,550 in autoantibody formation. Yamamoto et al. [27] showed that disease activity in patients with rheumatoid arthritis with insufficient response to methotrexate and patients with SLE decreased with CP-690,550 treatment, and that the elevated titers of both rheumatoid factor and anti-DNA antibodies became negative.
A crucial pathological feature of LN is the inflammatory infiltration triggered by tissue immune deposition [28,29]. Our results show a prominent infiltration of T cells and macrophages in renal tissue in vehicle-treated animals. Also, there was an increase in serum MCP-1, as well as prominent renal gene expression of regulated on activation, normal T cell expressed and secreted (RANTES) and MCP-1, which are potent chemoattractants for monocytes and T cells [30]. CP-690,550 treatment significantly reduced both T-cell and macrophage kidney infiltration, with a slight reduction in serum MCP-1 but a greater reduction in MCP-1 and RANTES renal gene expression. Finally, the renal reduction of all STAT components, indicating local inhibition of the downstream JAK-STAT pathway, may participate in the modulation of the renal inflammatory microenvironment. Some other authors have demonstrated that treatment of lupus-prone mice with JAK2 inhibitors led to disease prevention or improvement. Wang et al. showed that JAK2 inhibition in MRL/lpr mice led to a decrease in proteinuria, serum levels of dsDNA, renal cell infiltration, and deposition of IgG and C3 in the kidney [31]. Lu et al. used another JAK2 inhibitor in NZB/WF1 mice and showed improved survival, reduced proteinuria, and diminished dsDNA antibodies and spleen plasma cells; additionally, several cytokines were downregulated with treatment [32]. However, all of these studies were focused on the inhibition of JAK2; JAK2 specifically mediates cytokine signaling for red blood cells and platelets, and its inhibition causes anemia and low platelets [33,34]. The results of our present study clearly show that selective inhibition of JAK3 is ready to use as a new treatment for LN in clinical practice, given its profile and good tolerability in autoimmune diseases.
Apart from conventional inflammatory cytokines, gene expression analysis revealed the reduction of other mediators in renal tissue. TLR9 was, as expected, increased in control animals, and it was significantly reduced by all treatments. We also observed that the JAK-STAT pathway affected CD40L expression; blocking this pathway induced a severe and significant reduction of its expression, thereby affecting CD40-CD40L interaction. It is well known that this dyad plays a central role in the development of immune-mediated inflammatory disease processes [35,36], and we previously showed that blocking this pathway was also effective in the prevention of LN [37].
Finally, in our present study, we observed a dramatic drop in circulating levels of IL-12, IFN-α, and TNF-α in animals that received CP-690,550 treatment. The influence of JAK3 inhibition on inflammatory cytokines was also observed by Ghoreschi et al. [20], who reported a rapid improvement of the disease with CP-690,550 in a model of established arthritis, with inhibition of inflammatory mediators such as IFN-γ, IL-6, IL-12, and IL-23 in joint tissue. Further, Rosengren et al. showed that CP-690,550 decreased TNF-induced chemokine expression in fibroblasts from patients with rheumatoid arthritis [38]. No previous information has been reported regarding the effects of JAK inhibitors on IL-17 in experimental LN, but in our present study CP-690,550 treatment resulted in a consistent reduction of circulating IL-17 levels. In fact, the STAT3 transcription factor is essential for the differentiation of Th17 cells [7], and it is effectively inhibited by CP-690,550. As circulating IL-17A has been associated with immune complex deposition and complement activation in kidneys in a mouse model of lupus [8], it is quite likely that reduction of IL-17 in the bloodstream has a systemic mechanism that participates in the reduction of renal inflammation.
Conclusions
Our data strongly support the idea that the JAK-STAT pathway is implicated in the progression of renal inflammation in NZB/WF1 mice. Our results also provide evidence of the potential therapeutic effects of targeting this pathway with CP-690,550 as part of a robust strategy of immune disruption in autoimmune LN disease, particularly when treatment is initiated during the early phases of the disease. This has the potential to become a powerful new therapeutic tool in the treatment of LN and other autoimmune diseases. | 2018-03-23T11:11:30.635Z | 2016-07-01T00:00:00.000 | {
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