Engineering the Staple Oil Crop Brassica napus Enriched with α-Linolenic Acid Using the Perilla FAD2-FAD3 Fusion Gene.

Modern people generally suffer from α-linolenic acid (ALA) deficiency, since most staple food oils are low in ALA content. Thus, the enhancement of ALA in staple oil crops is of importance. In this study, the FAD2 and FAD3 coding regions from the ALA-king species Perilla frutescens were fused using a newly designed double linker LP4-2A, driven by a seed-specific promoter PNAP, and engineered into a rapeseed elite cultivar ZS10 with canola quality background. The mean ALA content in the seed oil of PNAP:PfFAD2-PfFAD3 (N23) T5 lines was 3.34-fold that of the control (32.08 vs 9.59%), with the best line being up to 37.47%. There are no significant side effects of the engineered constructs on the background traits including oil content. In fatty acid biosynthesis pathways, the expression levels of structural genes as well as regulatory genes were significantly upregulated in N23 lines. On the other hand, the expression levels of genes encoding the positive regulators of flavonoid-proanthocyanidin biosynthesis but negative regulators of oil accumulation were significantly downregulated. Surprisingly, the ALA level in PfFAD2-PfFAD3 transgenic rapeseed lines driven by the constitutive promoter PD35S was not increased or even showed a slight decrease due to the lower level of foreign gene expression and downregulation of the endogenous orthologous genes BnFAD2 and BnFAD3.

A systematic review and meta-analysis of long term physical and mental sequelae of COVID-19 pandemic: call for research priority and action.

The long-term physical and mental sequelae of COVID-19 are a growing public health concern, yet there is considerable uncertainty about their prevalence, persistence and predictors. We conducted a comprehensive, up-to-date meta-analysis of survivors' health consequences and sequelae for COVID-19. PubMed, Embase and the Cochrane Library were searched through Sep 30th, 2021. Observational studies that reported the prevalence of sequelae of COVID-19 were included. Two reviewers independently undertook the data extraction and quality assessment. Of the 36,625 records identified, a total of 151 studies were included involving 1,285,407 participants from thirty-two countries. At least one sequelae symptom occurred in 50.1% (95% CI 45.4-54.8) of COVID-19 survivors for up to 12 months after infection. The most common investigation findings included abnormalities on lung CT (56.9%, 95% CI 46.2-67.3) and abnormal pulmonary function tests (45.6%, 95% CI 36.3-55.0), followed by generalized symptoms, such as fatigue (28.7%, 95% CI 21.0-37.0), psychiatric symptoms (19.7%, 95% CI 16.1-23.6) mainly depression (18.3%, 95% CI 13.3-23.8) and PTSD (17.9%, 95% CI 11.6-25.3), and neurological symptoms (18.7%, 95% CI 16.2-21.4), such as cognitive deficits (19.7%, 95% CI 8.8-33.4) and memory impairment (17.5%, 95% CI 8.1-29.6). Subgroup analysis showed that participants with a higher risk of long-term sequelae were older, mostly male, living in a high-income country, with more severe status at acute infection. Individuals with severe infection suffered more from PTSD, sleep disturbance, cognitive deficits, concentration impairment, and gustatory dysfunction. Survivors with mild infection had high burden of anxiety and memory impairment after recovery. Our findings suggest that after recovery from acute COVID-19, half of survivors still have a high burden of either physical or mental sequelae up to at least 12 months. It is important to provide urgent and appropriate prevention and intervention management to preclude persistent or emerging long-term sequelae and to promote the physical and psychiatric wellbeing of COVID-19 survivors.

Burnout, emotional distress and sleep quality among Chinese psychiatric healthcare workers during the COVID-19 pandemic: a follow-up study.

Background: Different from the very early stages of the COVID-19 pandemic, burnout and chronic mental health problems among health care workers (HCWs) has become a challenge. Research is lacking on the relationship between burnout, stress, emotional distress and sleep quality. Methods: The Chinese center has been involved in the Cope-Corona project since the second survey (T2). Named after the project, a total of three cross-sectional surveys were distributed: T2 (February 16-20, 2021), T3 (May 10-14, 2022), and T4 (December 20-24, 2022). Burnout, depression, anxiety, sleep quality, workplace factors and individual resources were measured. Using the T4 data, we conducted structural equation model (SEM) to examine the mediating role of burnout in predicting emotional distress and sleep quality. Results: 96, 124, and 270 HCWs were enrolled at T2, T3, and T4, respectively. In line with the epidemic trends, the level of perceived COVID-19 related risks was significantly higher at T4, while the feeling of health and safety decreased significantly. At T4, the percentages of participants with clinically significant levels of depression and anxiety symptoms were 18.9% (51/270) and 9.3% (25/270), respectively, while 30.4% (82/270) of them reported poor or very poor sleep quality. According to the SEM, individual resources and workplace factors mainly had an indirect effect in predicting depression and anxiety via burnout. However, neither burnout nor stress was a mediator or predictor of sleep quality. Instead, individual resources, positive workplace factors, and younger age had a direct effect in predicting good sleep quality. Conclusion: Measures designed to enhance workplace factors and individual resources should be implemented to improve psychosomatic wellbeing of HCWs.

Workplace factors can predict the stress levels of healthcare workers during the COVID-19 pandemic: First interim results of a multicenter follow-up study.

Background: Research is lacking on the long-term influence of workplace factors on the mental health of health care workers during the COVID-19 pandemic. Methods: We distributed two online surveys to health care workers between May and October 2020 (T1) and between February and April 2021 (T2). Perceived stress, coronavirus-related risks, and workplace factors were measured via self-report questionnaires at both time points. We conducted hierarchical linear regression to investigate the predictive factors for high stress. Results: A total of 2,110 participants from seven countries and 4,240 participants from nine countries were enrolled at T1 and T2, respectively. Among them, 612 participated in both surveys. We called this cohort T1 + T2. High stress was reported in 53.8 and 61.6% of participants at T1 and T2, respectively. In cohort T1 + T2, compared with the baseline, the level of stress rose significantly (6.0 ± 2.9 vs. 6.4 ± 3.1), as did health/safety in the workplace (3.9 ± 0.8 vs. 4.2 ± 0.7). Unfortunately, we did not detect any significant difference concerning support in the workplace. Among all factors at baseline, being older than 35 [ß (95% CI) = -0.92 (-1.45, -0.40)], support [-0.80 (-1.29, -0.32)], and health/safety in the workplace [-0.33 (-0.65, -0.01)] were independent protective factors, while a positive history of mental disorders [0.81 (0.26, 1.37)] and rejection in private life [0.86 (0.48, 1.25)] were risk factors for high stress at T2. Conclusion: To relieve the high stress of health care workers, organizational-level approaches should be implemented, especially measures designed to enhance support, health/safety in the workplace, and to reduce the rejection of the public.

A systematic review and meta-analysis on prevalence of and risk factors associated with depression, anxiety and insomnia in infectious diseases, including COVID-19: a call to action.

Infectious disease epidemics have become more frequent and more complex during the 21st century, posing a health threat to the general public and leading to psychological symptoms. The current study was designed to investigate the prevalence of and risk factors associated with depression, anxiety and insomnia symptoms during epidemic outbreaks, including COVID-19. We systematically searched the PubMed, Embase, Web of Science, OVID, Medline, Cochrane databases, bioRxiv and medRxiv to identify studies that reported the prevalence of depression, anxiety or insomnia during infectious disease epidemics, up to August 14th, 2020. Prevalence of mental symptoms among different populations including the general public, health workers, university students, older adults, infected patients, survivors of infection, and pregnant women across all types of epidemics was pooled. In addition, prevalence of mental symptoms during COVID-19 was estimated by time using meta-regression analysis. A total of 17,506 papers were initially retrieved, and a final of 283 studies met the inclusion criteria, representing a total of 948,882 individuals. The pooled prevalence of depression ranged from 23.1%, 95% confidential intervals (95% CI: [13.9-32.2]) in survivors to 43.3% (95% CI: [27.1-59.6]) in university students, the pooled prevalence of anxiety ranged from 25.0% (95% CI: [12.0-38.0]) in older adults to 43.3% (95% CI: [23.3-63.3]) in pregnant women, and insomnia symptoms ranged from 29.7% (95% CI: [24.4-34.9]) in the general public to 58.4% (95% CI: [28.1-88.6]) in university students. Prevalence of moderate-to-severe mental symptoms was lower but had substantial variation across different populations. The prevalence of mental problems increased over time during the COVID-19 pandemic among the general public, health workers and university students, and decreased among infected patients. Factors associated with increased prevalence for all three mental health symptoms included female sex, and having physical disorders, psychiatric disorders, COVID infection, colleagues or family members infected, experience of frontline work, close contact with infected patients, high exposure risk, quarantine experience and high concern about epidemics. Frequent exercise and good social support were associated with lower risk for these three mental symptoms. In conclusion, mental symptoms are common during epidemics with substantial variation across populations. The population-specific psychological crisis management are needed to decrease the burden of psychological problem and improve the mental wellbeing during epidemic.

Efficacy and practice of facemask use in general population: a systematic review and meta-analysis.

In recent decades, respiratory infections, including SARS, HINI and the currently spreading COVID-19, caused by various viruses such as influenza and coronavirus have seriously threatened human health. It has generated inconsistent recommendations on the mandatory use of facemasks across countries on a population level due to insufficient evidence on the efficacy of facemask use among the general population. This meta-analysis aimed to explore (1) the efficacy of facemask use on preventing respiratory infections, and (2) the perceptions, intentions, and practice about facemask use among the general population worldwide. We searched PubMed, MEDLINE, Web of Science, Cochrane, bioRxiv, and medRxiv databases since inception to August 17, 2020. From 21,341 records identified, eight RCTs on facemask in preventing infections and 78 studies on perception, intention, and practice of facemask use among the general population were included in the analysis. The meta-analysis of RCTs found a significant protective effect of facemask intervention (OR = 0.84; 95% CI = 0.71-0.99; I2 = 0%). This protective effect was even more pronounced when the intervention duration was more than two weeks (OR = 0.76; 95% CI = 0.66-0.88; I2 = 0%). The meta-analysis of observational studies on perception, intention, and practice on facemask use showed that 71% of respondents perceived facemasks to be effective for infection prevention, 68% of respondents would wear facemasks, and 54% of respondents wore facemasks for preventing respiratory infections. Differences in perception, intention, and practice behavior of facemask use in different regions may be related to the impact of respiratory infections, regional culture, and policies. The governments and relevant organizations should make effort to reduce the barriers in the use of facemasks.

A systematic review and meta-analysis on the prevalence of stigma in infectious diseases, including COVID-19: a call to action.

Infectious diseases, including COVID-19, are crucial public health issues and may lead to considerable fear among the general public and stigmatization of, and discrimination against, specific populations. This meta-analysis aimed to estimate the pooled prevalence of stigma in infectious disease epidemics. We systematically searched PubMed, PsycINFO, Embase, MEDLINE, Web of Science, and Cochrane databases since inception to June 08, 2021, and reported the prevalence of stigma towards people with infectious diseases including SARS, H1N1, MERS, Zika, Ebola, and COVID-19. A total of 50 eligible articles were included that contributed 51 estimates of prevalence in 92722 participants. The overall pooled prevalence of stigma across all populations was 34% [95% CI: 28-40%], including enacted stigma (36% [95% CI: 28-44%]) and perceived stigma (31% [95% CI: 22-40%]). The prevalence of stigma in patients, community population, and health care workers, was 38% [95% CI: 12- 65%], 36% [95% CI: 28-45%], and 30% [95% CI: 20-40%], respectively. The prevalence of stigma in participants from low- and middle-income countries was 37% [95% CI: 29-45%], which is higher than that from high-income countries (27% [95% CI: 18-36%]) though this difference was not statistically significant. A similar trend of prevalence of stigma was also observed in individuals with lower education (47% [95% CI: 23-71%]) compared to higher education level (33% [95% CI: 23-4%]). These findings indicate that stigma is a significant public health concern, and effective and comprehensive interventions are needed to counteract the damaging effects of the infodemics during infectious disease epidemics, including COVID-19, and reduce infectious disease-related stigma.

Cognitive improvement among abstinent methamphetamine users: A 2-year prospective longitudinal study.

BACKGROUND AND OBJECTIVES: The adverse impact of chronic methamphetamine (MA) use on cognitive function has been described in previous studies, but limited evidence is available for abstinent users from prospective longitudinal studies. The aim of the present study was to assess cognitive function of varying abstinent duration. METHODS: This prospective longitudinal study was conducted with baseline and four follow-up interviews every 6 months over 2 years in 358 MA users in Guangdong province, China. The Montreal Cognitive Assessment (MoCA) was used to measure cognitive function. Generalized estimating equation (GEE) analysis was used to examine within-subjects relationships between abstinence and cognitive consequences over time. RESULTS: The repeated measure analysis of variance showed significant differences in the total MoCA score and all subscale scores (except Orientation) in the 24 months follow-up. The GEE model showed that abstinence from MA in the past 6 months predicted an increase of 0.66 (95% confidence interval [CI] = 0.29 to 1.05, p = .002) in MoCA score changes compared with the nonabstinence MA users. Abstinence in the past 12, 18, and 24 months predicted an increase in MoCA total score changes of 1.25 (95% CI = -0.23 to 2.74), 2.15 (95% CI = -0.79 to 5.09), and 5.28 (95% CI = -2.01 to 12.58), respectively, but none of these was statistically significant. DISCUSSION AND CONCLUSIONS: Cognitive function was potentially improved following 6 months of MA abstinence. SCIENTIFIC SIGNIFICANCE: This study extends prior research by long-term follow-up in big sample MA abstinence users. Findings from study support the need for a comprehensive measure to decrease MA use and promote the recovery of cognitive impairment.

Identifying Modifiable Risk Factors for Relapse in Patients With Schizophrenia in China.

BACKGROUND: Preventing relapse of schizophrenic patients is really a challenge. The present study sought to provide more explicit evidence and factors of different grades and weights by a series of step-by-step analysis through χ2 test, logistic regression analysis and decision-tree model. The results of this study may contribute to controlling relapse of schizophrenic patients. METHODS: A total of 1,487 schizophrenia patients were included who were 18-65 years of age and discharged from 10 hospitals in China from January 2009 to August 2009 and from September 2011 to February 2012 with improvements or recovery of treatment effect. We used a questionnaire to collect information about relapse and correlative factors during one year after discharge by medical record collection and telephone interview. The χ2 test and logistic regression analysis were used to identify risk factors and high-risk factors firstly, and then a decision-tree model was used to find predictive factors. RESULTS: The χ2 test found nine risk factors which were associated with relapse. Logistic regression analysis also showed four high-risk factors further (medication adherence, occupational status, ability of daily living, payment method of medical costs). At last, a decision-tree model revealed four predictors of relapse; it showed that medication adherence was the first grade and the most powerful predictor of relapse (relapse rate for adherence vs. nonadherence: 22.9 vs. 55.7%, χ2 = 116.36, p < 0.001). The second grade factor was occupational status (employment vs. unemployment: 19.7 vs. 42.7%, χ2 = 17.72, p < 0.001); the third grade factors were ability of daily living (normal vs. difficult: 28.4 vs. 54.3%, χ2 = 8.61, p = 0.010) and household income (household income ≥ 3000 RMB vs. <3000 RMB: 28.6 vs. 42.4%, χ2 = 6.30, p = 0.036). The overall positive predictive value (PPV) of the logistic regression was 0.740, and the decision-tree model was 0.726. Both models were reliable. CONCLUSIONS: For schizophrenic patients discharged from hospital, who had good medication adherence, more higher household income, be employed and normal ability of daily living, would be less likely to relapse. Decision tree provides a new path for doctors to find the schizophrenic inpatient's relapse risk and give them reasonable treatment suggestions after discharge.

Effects of agomelatine and mirtazapine on sleep disturbances in major depressive disorder: evidence from polysomnographic and resting-state functional connectivity analyses.

To investigate effects of agomelatine and mirtazapine on sleep disturbances in patients with major depressive disorder. A total of 30 depressed patients with sleep disturbances, 27 of which completed the study, took agomelatine or mirtazapine for 8 weeks. Subjective scales were administered, and polysomnography was performed at baseline and at the end of week 1 and 8. Functional magnetic resonance imaging was performed at baseline and at the end of week 8. Compared with baseline, scores on the Hamilton Depression Scale, Hamilton Anxiety Scale, Pittsburgh Sleep Quality Index, Sleep Dysfunction Rating Scale, and Insomnia Severity Index after 8 weeks of treatment significantly decreased in both groups, with no significant differences between groups, accompanied by significant increases in total sleep time, sleep efficiency, and rapid eye movement (REM) sleep and significant decrease in wake after sleep onset. Mirtazapine treatment increased N3 sleep at week 1 compared with agomelatine treatment, but this difference disappeared at week 8. The increases in the percentage and duration of N3 sleep were positively correlated with increases in connectivity between right dorsal lateral prefrontal cortex (dlPFC) and right precuneus and between left posterior cingulate cortex and right precuneus in both groups, respectively. Functional connectivity (FC) between right dlPFC and left precuneus in mirtazapine group was higher compared with agomelatine group after 8 weeks of treatment. These findings indicated that both agomelatine and mirtazapine improved sleep in depressed patients, and the effect of mirtazapine was greater than agomelatine with regard to rapidly increasing N3 sleep and gradually improving FC in the brain.

Objective sleep duration is associated with cognitive deficits in primary insomnia: BDNF may play a role.

Study Objectives: Objective sleep duration has been linked to insomnia severity. However, cognitive functions of people with insomnia with different sleep durations have been seldom addressed. Brain-derived neurotrophic factor (BDNF) has an important role in cognitive function and has been linked to clinical insomnia recently. The present study aimed to evaluate the comprehensive cognitive functions in people with primary insomnia with different objective sleep durations, and further examine the involvement of peripheral BDNF. Methods: Fifty-seven people with insomnia were subdivided into short sleep duration (SSD, sleep time < 6 hr) group and normal sleep duration (NSD, sleep time ≥ 6 hr) group based on polysomnography data. Twenty-nine healthy controls (HC) were matched on age, gender, and education. Cognitive function was assessed using a comprehensive and sensitive neuropsychological test battery. Both objective and subjective insomnia statuses were estimated. Serum BDNF level was measured using enzyme-linked immune sorbent assay. Results: Compared with HC, the SSD group showed impaired neuropsychological performances in spatial span, brief visuospatial memory test, fluency, managing emotions, and continuous performance tests. In contrast, NSD had bad performance only in brief visuospatial memory test and continuous performance tests, and relatively better than SSD group in the latter test. People with SSD insomnia but not NSD had decreased BDNF levels compared with HC, and neuropsychological performance was positively correlated with BDNF levels only in SSD group. Conclusions: Primary insomnia was associated with impaired neuropsychological performance, and the impairment might be related to decreased objective sleep duration. In addition, decreased peripheral BDNF might mediate the impaired cognitive functions of people with insomnia with SSD.

Erratum: Electroconvulsive therapy-induced brain functional connectivity predicts therapeutic efficacy in patients with schizophrenia: a multivariate pattern recognition study.

[This corrects the article DOI: 10.1038/s41537-017-0023-7.].

Altered Brain Network Connectivity as a Potential Endophenotype of Schizophrenia.

Abnormal functional brain connectivity could be considered an endophenotype of psychosis in schizophrenia. Identifying candidate endophenotypes may serve as a tool for elucidating its biological and neural mechanisms. The present study investigated the similarities and differences of features of brain network connectivity between patients and their first-degree relatives. Independent component analysis was conducted on imaging data collected from 34 healthy controls, 33 schizophrenia patients, and 30 unaffected first-degree relatives. The correlation between functional connectivity with neurocognitive performance and clinical symptoms were calculated. Abnormalities of between-network connectivity largely overlapped in patients and first-degree relatives, but the extent of such abnormalities was relatively minor in relatives. Negative connectivity between language networks and executive control networks was impaired in schizophrenia patients and their first-degree relatives, and this decreased connectivity was correlated with performance in language processing. Similar impairments were found in high-visual network and executive network coupling, and this decreased connection was correlated with the severity of positive symptoms in patients. The results indicated that abnormal functional connectivity within and between perceptual systems (i.e., high-visual and language) and executive control networks was related to the generic risk of schizophrenia, which makes it a potential endophenotype for schizophrenia.

Electroconvulsive therapy-induced brain functional connectivity predicts therapeutic efficacy in patients with schizophrenia: a multivariate pattern recognition study.

Previous studies suggested that electroconvulsive therapy can influence regional metabolism and dopamine signaling, thereby alleviating symptoms of schizophrenia. It remains unclear what patients may benefit more from the treatment. The present study sought to identify biomarkers that predict the electroconvulsive therapy response in individual patients. Thirty-four schizophrenia patients and 34 controls were included in this study. Patients were scanned prior to treatment and after 6 weeks of treatment with antipsychotics only (n = 16) or a combination of antipsychotics and electroconvulsive therapy (n = 13). Subject-specific intrinsic connectivity networks were computed for each subject using a group information-guided independent component analysis technique. Classifiers were built to distinguish patients from controls and quantify brain states based on intrinsic connectivity networks. A general linear model was built on the classification scores of first scan (referred to as baseline classification scores) to predict treatment response. Classifiers built on the default mode network, the temporal lobe network, the language network, the corticostriatal network, the frontal-parietal network, and the cerebellum achieved a cross-validated classification accuracy of 83.82%, with specificity of 91.18% and sensitivity of 76.47%. After the electroconvulsive therapy, psychosis symptoms of the patients were relieved and classification scores of the patients were decreased. Moreover, the baseline classification scores were predictive for the treatment outcome. Schizophrenia patients exhibited functional deviations in multiple intrinsic connectivity networks which were able to distinguish patients from healthy controls at an individual level. Patients with lower classification scores prior to treatment had better treatment outcome, indicating that the baseline classification scores before treatment is a good predictor for treatment outcome.

Pattern and related factors of cognitive impairment among chronic methamphetamine users.

BACKGROUND AND OBJECTIVES: Methamphetamine (MA) use is increasingly prevalent in East and Southeast Asia and commonly associated with cognitive impairment. The present study estimated the characteristics of cognitive impairment and explored the associated potential factors among chronic MA users. METHODS: The data were from the baseline visit of a longitudinal study among synthetic drug users. The baseline survey was conducted in detoxification and rehabilitation centers in Guangdong province, China, from September to December in 2013. A total of 528 participants were included in our analysis. Cognitive impairment was measured by the Montreal Cognitive Assessment (MoCA). Logistic regression was performed to explore the risk factors associated with cognitive impairment. RESULTS: Approximately 69.89% of the study participants exhibited cognitive impairment according to MoCA scores. Multiple logistic regression analyses indicated that older age (≥30 years old), a longer duration of MA use (>24 months), and a higher frequency of MA use (everyday) were associated with cognitive impairment, with adjusted odds ratios (ORs) of 1.58 (95% confidence interval [CI]: 1.07-2.34), 1.53 (95%CI: 1.01-2.31), and 1.55 (95%CI: 1.05-2.30), respectively. Methamphetamine users that had a higher level of education had a lower risk of cognitive impairment(OR = .59; 95%CI: .38-.93). CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: Cognitive impairment occurred frequently among chronic MA users. The causal relationship between cognitive impairment and MA use needs to be ascertained in longitudinal studies in future work. Our study provides evidence for the development of intervention strategies for the prevention of MA use and associated cognitive impairment. (Am J Addict 2017;26:145-151).

Insomnia and Multimorbidity in the Community Elderly in China.

STUDY OBJECTIVES: To examine the comorbidity between insomnia and medical conditions. METHODS: This cross-sectional study was conducted in community adults, aged ≥ 60 years, who resided in one of four major cities in northern China. Sociodemographic and clinical data were collected simultaneously. A total of 3,176 elderly adults (1,292 male, mean ± standard deviation age 70.2 ± 6.8 years; 1,884 female, 68.8 ± 6.7 years) were interviewed. RESULTS: The prevalence of specific medical conditions in both people with insomnia and people without insomnia was detected. Significantly higher proportions of arrhythmia, hypertension, cerebral hemorrhage, migraine, and hyperlipidemia were observed in people with insomnia than in people without insomnia. Moreover, a significantly higher proportion of insomnia was seen in elderly people with arrhythmia and migraine. We also found that elderly people with insomnia who took sleep medications reported a higher prevalence of coexisting arrhythmia, hypertension, and migraine, even after adjusting for age, sex, and depression. CONCLUSIONS: Our results indicate associations between insomnia and medical conditions in the community elderly in China. People who complained of insomnia had poorer physical health conditions. Sleep medication may not be a covariate that influences the comorbidity of some specific physical conditions. CLINICAL TRIAL REGISTRATION: Trial name: The study of diagnosis and treatment of senile dementia in Hebei Province; URL: http://www.chictr.org.cn/showproj.aspx?proj=8194; Registration number: ChiCTR-RRC-11001345.