Anti-SFT2D2 autoantibodies alter dendrite spine and cause psychotic behavior in mice.

BACKGROUND: Autoimmunity plays an important role in schizophrenia (SCZ). Autoantibodies against SFT2D2 have been reported in patients with SCZ; however, the specific mechanism remains unclear. This study aimed to describe an autoimmune model, namely, mice immunized against SFT2D2-peptides. METHODS: ApoE-/- and WT mice (C57BL/6) were immunized four times (day 0, day 14, day 21, day 35) with SFT2D2 peptide or KLH via subcutaneous injection. Behavioral tests were conducted after the third immunization, and immunochemistry of brain tissue were performed after the sacrifice of the mice. RESULTS: Active immunization with KLH-coupled SFT2D2-derived peptides in both WT and ApoE-/- (compromised blood-brain barrier) mice led to high circulating levels of anti-SFT2D2 IgG. While there was no detectable deficit in WT mice, impaired pre-pulse inhibition, motor impairments, and reduced cognition in ApoE-/- mice, without signs of anxiety and depression were observed. In addition, immunohistochemical assays demonstrated that activated microglia and astrocytes were increased but neuronal dendritic spine densities were decreased, accompanied by increased expression of complement molecule C4 across brain regions in ApoE-/- mice. CONCLUSIONS: In model mice with compromised blood-brain barrier, endogenous anti-SFT2D2 IgG can activate glial cells and modulate synaptic plasticity, and induce a series of psychosis-like changes. These antibodies may reveal valuable therapeutic targets, which may improve the treatment strategies for a subgroup of SCZ patients.

Genetic Screening of Targeted Region on the Chromosome 22q11.2 in Patients with Microtia and Congenital Heart Defect.

Microtia is a congenital malformation characterized by a small, abnormally shaped auricle (pinna) ranging in severity. Congenital heart defect (CHD) is one of the comorbid anomalies with microtia. However, the genetic basis of the co-existence of microtia and CHD remains unclear. Copy number variations (CNVs) of 22q11.2 contribute significantly to microtia and CHD, respectively, thus suggesting a possible shared genetic cause embedded in this genomic region. In this study, 19 sporadic patients with microtia and CHD, as well as a nuclear family, were enrolled for genetic screening of single nucleotide variations (SNVs) and CNVs in 22q11.2 by target capture sequencing. We detected a total of 105 potential deleterious variations, which were enriched in ear- or heart-development-related genes, including TBX1 and DGCR8. The gene burden analysis also suggested that these genes carry more deleterious mutations in the patients, as well as several other genes associated with cardiac development, such as CLTCL1. Additionally, a microduplication harboring SUSD2 was validated in an independent cohort. This study provides new insights into the underlying mechanisms for the comorbidity of microtia and CHD focusing on chromosome 22q11.2, and suggests that a combination of genetic variations, including SNVs and CNVs, may play a crucial role instead of single gene mutation.

Elevated BICD2 DNA methylation in blood of major depressive disorder patients and reduction of depressive-like behaviors in hippocampal Bicd2-knockdown mice.

Major depressive disorder (MDD) is a prevalent and devastating mental illness. To date, the diagnosis of MDD is largely dependent on clinical interviews and questionnaires and still lacks a reliable biomarker. DNA methylation has a stable and reversible nature and is likely associated with the course and therapeutic efficacy of complex diseases, which may play an important role in the etiology of a disease. Here, we identified and validated a DNA methylation biomarker for MDD from four independent cohorts of the Chinese Han population. First, we integrated the analysis of the DNA methylation microarray (n = 80) and RNA expression microarray data (n = 40) and identified BICD2 as the top-ranked gene. In the replication phase, we employed the Sequenom MassARRAY method to confirm the DNA hypermethylation change in a large sample size (n = 1,346) and used the methylation-sensitive restriction enzymes and a quantitative PCR approach (MSE-qPCR) and qPCR method to confirm the correlation between DNA hypermethylation and mRNA down-regulation of BICD2 (n = 60). The results were replicated in the peripheral blood of mice with depressive-like behaviors, while in the hippocampus of mice, Bicd2 showed DNA hypomethylation and mRNA/protein up-regulation. Hippocampal Bicd2 knockdown demonstrates antidepressant action in the chronic unpredictable mild stress (CUMS) mouse model of depression, which may be mediated by increased BDNF expression. Our study identified a potential DNA methylation biomarker and investigated its functional implications, which could be exploited to improve the diagnosis and treatment of MDD.

Adaptive Recognition of Bioacoustic Signals in Smart Aquaculture Engineering Based on r-Sigmoid and Higher-Order Cumulants.

In recent years, interest in aquaculture acoustic signal has risen since the development of precision agriculture technology. Underwater acoustic signals are known to be noisy, especially as they are inevitably mixed with a large amount of environmental background noise, causing severe interference in the extraction of signal features and the revelation of internal laws. Furthermore, interference adds a considerable burden on the transmission, storage, and processing of data. A signal recognition curve (SRC) algorithm is proposed based on higher-order cumulants (HOC) and a recognition-sigmoid function for feature extraction of target signals. The signal data of interest can be accurately identified using the SRC. The analysis and verification of the algorithm are carried out in this study. The results show that when the SNR is greater than 7 dB, the SRC algorithm is effective, and the performance improvement is maximized when the SNR is 11 dB. Furthermore, the SRC algorithm has shown better flexibility and robustness in application.

The SFT2D2 gene is associated with the autoimmune pathology of schizophrenia in a Chinese population.

Background: The relative risk of GWAS-confirmed loci strongly associated with schizophrenia may be underestimated due to the decay of linkage disequilibrium between index SNPs and causal variants. This study is aimed to investigate schizophrenia-associated signals detected in the 1q24-25 region in order to identify a causal variant in LD with GWAS index SNPs, and the potential biological functions of the risk gene. Methods: Re-genotyping analysis was performed in the 1q24-25 region that harbors three GWAS index SNPs associated with schizophrenia (rs10489202, rs11586522, and rs6670165) in total of 9801 case-control subjects of Chinese Han origin. Circulating autoantibody levels were assessed using an in-house ELISA against a protein derived fragment encoded by SFT2D2 in total of 682 plasma samples. Results: A rare variant (rs532193193) in the SFT2D2 locus was identified to be strongly associated with schizophrenia. Compared with control subjects, patients with schizophrenia showed increased anti-SFT2D2 IgG levels. Receiver operating characteristic (ROC) analysis revealed an area under the ROC curve (AUC) of 0.803 with sensitivity of 28.57% against specificity of 95% for the anti-SFT2D2 IgG assay. Discussion: Our findings indicate that SFT2D2 is a novel gene for risk of schizophrenia, while endogenous anti-SFT2D2 IgG may underlie the pathophysiology of the immunological aspects of schizophrenia.

Medical care of rare and undiagnosed diseases: Prospects and challenges.

Rare and undiagnosed diseases tend to be diverse, misdiagnosed, and difficult to diagnose. In some cases, the disease is progressive and life-threatening. Yet, to date, an estimated 95% of rare diseases have no approved therapy. Therefore, rare and undiagnosed diseases are considered the ultimate challenges for understanding human diseases. Here, we review the research progress, research frontiers, and important scientific issues related to rare and undiagnosed diseases. We mainly focus on five topics: (1) the identification and functional analysis of disease-causing genes; (2) the construction of cells, organoids, and animal models for mechanism validation; (3) subtyping and diagnosis; (4) treatment and drug screening based on causative genes and mutations; and (5) new technologies and methods for studying rare and undiagnosed diseases. In this review, we briefly update and discuss the pathogenic mechanisms and precision medicine for rare and undiagnosed diseases.

Fat mass and obesity-associated protein regulates RNA methylation associated with depression-like behavior in mice.

Post-transcriptional modifications of RNA, such as RNA methylation, can epigenetically regulate behavior, for instance learning and memory. However, it is unclear whether RNA methylation plays a critical role in the pathophysiology of major depression disorder (MDD). Here, we report that expression of the fat mass and obesity associated gene (FTO), an RNA demethylase, is downregulated in the hippocampus of patients with MDD and mouse models of depression. Suppressing Fto expression in the mouse hippocampus results in depression-like behaviors in adult mice, whereas overexpression of FTO expression leads to rescue of the depression-like phenotype. Epitranscriptomic profiling of N6-methyladenosine (m6A) RNA methylation in the hippocampus of Fto knockdown (KD), Fto knockout (cKO), and FTO-overexpressing (OE) mice allows us to identify adrenoceptor beta 2 (Adrb2) mRNA as a target of FTO. ADRB2 stimulation rescues the depression-like behaviors in mice and spine loss induced by hippocampal Fto deficiency, possibly via the modulation of hippocampal SIRT1 expression by c-MYC. Our findings suggest that FTO is a regulator of a mechanism underlying depression-like behavior in mice.

Patterns and risk factors of peripherally inserted central venous catheter-related symptomatic thrombosis events in patients with malignant tumors receiving chemotherapy.

OBJECTIVE: Peripherally inserted central venous catheter (PICC)-related thrombosis (PRT) is a serious complication that can lead to interruptions in chemotherapy and other supportive care, as well as increased hospital stay and costs. We conducted a retrospective study to evaluate the patterns of symptomatic PRT in patients with cancer undergoing chemotherapy and their risk factors. METHODS: A retrospective study of 938 PICC patients from our institution between November 2014 and July 2017 was performed. Symptomatic PRT events were confirmed by color Doppler ultrasonography or computed tomography pulmonary angiography in the presence of clinical symptoms. The variables of interest were extracted from the electronic medical record system. Logistic regression analysis was used to determine the risk factors for PRT. RESULTS: Of the 938 patients who were followed up for more than 120,000 patient-days, 63 patients (6.7%; 0.51 per 1000 catheter-days) had symptomatic PRT. Sixty-one patients were diagnosed with upper extremity venous thrombosis (UEVT), of which 18 were isolated superficial vein thrombosis (SVT), 19 were isolated deep vein thrombosis (DVT), and 24 were extensive venous thrombosis (EVT). Two patients were diagnosed with pulmonary embolism, and two patients were diagnosed with UEVT with pulmonary embolism. The symptomatic SVT occurred in 42 of 938 patients with cancer (4.5%), which accounted for 68.9% of all UEVT events. The median time to PRT was 21 days, and the median time to catheter removal in the PRT group was 66 days as compared with 117 days in the no PRT group. Predictors associated with increased risk of PRT were age >60 years (odds ratio [OR], 2.142; 95% confidence interval [CI], 1.118-4.103) and a chemotherapy regimen containing fluorouracil (OR, 2.429; 95% CI, 1.013-5.825). Hypertension with medication was a protective factor for PRT (OR, 0.306; 95% CI, 0.113-0.828). Among the 28 patients who did not remove their PICCs immediately after PRT was diagnosed, patients with SVT, DVT, and EVT had similar success rates of retaining catheters in situ after anticoagulant therapy (SVT, 83.3%; DVT, 62.5%; EVT, 75.0%; P = .667). CONCLUSIONS: Age >60 years and chemotherapy regimens containing fluorouracil were independent risk factors for PRT and hypertension with medication was associated with a lower risk of PRT in patients with cancer with PICCs receiving chemotherapy. PICCs-related SVT was a frequent type of PRT, which might need a better understanding and anticoagulant therapy in patients with cancer with PICCs.

Clinical efficacy of irinotecan plus raltitrexed chemotherapy in refractory esophageal squamous cell cancer.

Our retrospective study assessed the efficacy and safety of irinotecan plus raltitrexed in esophageal squamous cell cancer (ESCC) patients who were previously treated with multiple systemic therapies. Between January 2016 and December 2018, records of 38 ESCC patients who underwent irinotecan plus raltitrexed chemotherapy after at least one line of chemotherapy were reviewed. Efficacy assessment was performed every two cycles according to the RECIST version 1.1. A total of 95 cycles of chemotherapy were administered, and the median course was 3 (range 2-6). There was no treatment-related death. Nine patients had partial response, 21 had stable disease and eight had progressive disease. The overall objective response rate was 23.68% (9/38) and the disease control rate was78.94% (30/38). After a median follow-up of 18.5 months, the median progression-free survival and overall survival were 105 and 221 days, respectively. There were five patients (13.15%) with grade 3/4 leukopenia, three patients (7.89%) with grade 3/4 neutropenia and one patient (2.63%) with grade 3/4 diarrhea. The combination of irinotecan plus raltitrexed was effective for pretreated ESCC patients. Further studies are needed to determine the optimal dose of the two drugs.

The benefit of taxane-based therapies over fluoropyrimidine plus platinum (FP) in the treatment of esophageal cancer: a meta-analysis of clinical studies.

PURPOSE: Fluoropyrimidine plus platinum (FP) is currently the standard treatment for esophageal cancer (EC). In recent years, taxane-based chemotherapy has also been used and has shown good efficacy in EC. This study aims to investigate the advantages of taxane-based over FP chemotherapy, as well as discuss its drawbacks, in the treatment of EC. PATIENTS AND METHODS: A literature search was done for studies comparing clinical outcomes between taxane-based and FP chemotherapy in EC. Pooled analyses were performed to compare the efficacy and grade 3/4 adverse events in patients who received neoadjuvant chemotherapy (NACT), neoadjuvant chemoradiotherapy (NACRT), or definitive chemoradiotherapy (dCRT). Subgroup analyses were also conducted in esophageal squamous cell carcinoma (ESCC). RESULTS: Thirty-one studies with a total of 3,912 patients were included in the analysis. Better long-term survival was found in patients who received taxane-based NACT (progression-free survival (PFS): pooled HR=0.58, P=0.0008; and overall survival (OS): pooled HR=0.50, P<0.00001) and dCRT (PFS: pooled HR=0.75, P<0.0001). In NACRT, taxane-based treatment and FP showed similar efficacy. In ESCC patients, taxane-based treatment showed better OS (NACT: pooled HR=0.57, P=0.02; NACRT: pooled HR=0.51, P=0.03; and dCRT: pooled HR=0.73, P<0.0001) than FP chemotherapy. Furthermore, taxane-based therapy also showed a better short-term response (complete response (CR), objective response rate (ORR), disease control rate (DCR), or pathologic complete response (pCR). However, taxane-based therapy was significantly correlated with a higher incidence of grade 3/4 leukopenia, neutropenia, and diarrhea. CONCLUSION: Compared to FP, taxane-based therapy produced better clinical response and outcomes in EC patients receiving NACT or dCRT, and in all types of therapy in patients with ESCC. Taxane-based treatment is associated with more frequent toxicity.

Population transcriptomic characterization of the genetic and expression variation of a candidate progenitor of Miscanthus energy crops.

The use of transcriptome data in the study of the population genetics of a species can capture faint signals of both genetic variation and expression variation and can provide a broad picture of a species' genomic response to environmental conditions. In this study, we characterized the genetic and expression diversity of Miscanthus lutarioriparius by comparing more than 16,225 transcripts obtained from 78 individuals, belonging to 10 populations distributed across the species' entire geographic range. We only observed a low level of nucleotide diversity (π = 0.000434) among the transcriptome data of these populations, which is consistent with highly conserved sequences of functional elements and protein-coding genes captured with this method. Tests of population divergence using the transcriptome data were consistent with previous microsatellite data but proved to be more sensitive, particularly if gene expression variation was considered as well. For example, the analysis of expression data showed that genes involved in photosynthetic processes and responses to temperature or reactive oxygen species stimuli were significantly enriched in certain populations. This differential gene expression was primarily observed among populations and not within populations. Interestingly, nucleotide diversity was significantly negatively correlated with expression diversity within populations, while this correlation was positive among populations. This suggests that genetic and expression variation play separate roles in adaptation and population persistence. Combining analyses of genetic and gene expression variation represents a promising approach for studying the population genetics of wild species and may uncover both adaptive and nonadaptive processes.

Haplotypes Phased from Population Transcriptomes Detecting Selection in the Initial Adaptation of Miscanthus lutarioriparius to Stressful Environments.

Adaptation is a characteristic that enhances the survival or reproduction of organisms; selection is the critical process leading to adaptive evolution. Therefore, detecting selection is important in studying evolutionary biology. Changes in allele frequency are fundamental to adaptive evolution. The allele frequency of entire genes at the genomic scale is more intensive and precise for analyzing selection effects, compared with simple sequence repeat and single nucleotide polymorphism (SNP) alleles from nuclear gene fragments. Here, we analyzed 29,094 SNPs derived from 80 individuals of 14 L. Liou ex S.L. Chen & Renvoize populations planted near their native habitat (Jiangxia, Hubei Province, JH) and a stressful environment (Qingyang, Gansu Province, QG) to detect selection during initial adaptation. The nucleotide diversity of over 60% of genes was decreased in QG compared with JH, suggesting that most genes were undergoing selection in the stressful environment. We explored a new approach based on haplotype data inferred from RNA-seq data to analyze the change in frequency between two sites and to detect selection signals. In total, 402 and 51 genes were found to be targets of positive and negative selection, respectively. Among these candidate genes, the enrichment of abiotic stress-response genes and photosynthesis-related genes might have been responsible for establishment in the stressful environment. This is the first study assessing the change in allele frequency at the genomic level during adaptation. The method in which allele frequency detects selection during initial adaptation using population RNA-seq data would be useful for developing evolutionary biology.

Systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change.

BACKGROUND: Long non-coding RNA (lncRNA) is a class of non-coding RNA with important regulatory roles in biological process of organisms. The systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change are still poorly understood. Here we identified 17,610 lncRNAs and calculated their expression levels based on RNA-seq of 80 individuals of Miscanthus lutarioriparius from two environments, the nearly native habitats and transplanted field, respectively. RESULTS: LncRNAs had significantly higher expression diversity and lower expression frequency in population than protein coding mRNAs in both environments, which suggested that lncRNAs may experience more relaxed selection or divergent evolution in population compared with protein coding RNAs. In addition, the increase of expression diversity for lncRNAs was always significantly higher and the magnitude of fold change of expression in new stress environment was significantly larger than protein-coding mRNAs. These results suggested that lncRNAs may be more sensitive to environmental change than protein-coding mRNAs. Analysis of environment-robust and environment-specific lncRNA-mRNA co-expression network between two environments revealed the characterization of lncRNAs in response to environmental change. Furthermore, candidate lncRNAs contributing to water use efficiency (WUE) identified based on the WUE-lncRNA-mRNA co-expression network suggested the roles of lncRNAs in response to environmental change. CONCLUSION: Our study provided a comprehensive understanding of expression characterization of lncRNAs in population for M. lutarioriparius under field condition, which would be useful to explore the roles of lncRNAs and could accelerate the process of adaptation in new environment for many plants.

Population transcriptomics uncovers the regulation of gene expression variation in adaptation to changing environment.

Expression variation plays an important role in plant adaptation, but little is known about the factors impacting the expression variation when population adapts to changing environment. We used RNA-seq data from 80 individuals in 14 Miscanthus lutarioriparius populations, which were transplanted into a harsh environment from native habitat, to investigate the expression level, expression diversity and genetic diversity for genes expressed in both environments. The expression level of genes with lower expression level or without SNP tended to be more changeable in new environment, which suggested highly expressed genes experienced stronger purifying selection than those at lower level. Low proportion of genes with population effect confirmed the weak population structure and frequent gene flow in these populations. Meanwhile, the number of genes with environment effect was the most frequent compared with that with population effect. Our results showed that environment and genetic diversity were the main factors determining gene expression variation in population. This study could facilitate understanding the mechanisms of global gene expression variation when plant population adapts to changing environment.

The Coordination of Gene Expression within Photosynthesis Pathway for Acclimation of C4 Energy Crop Miscanthus lutarioriparius.

As a promising candidate for the second-generation C4 energy crop, Miscanthus lutarioriparius has well acclimated to the water-limited and high-light Loess Plateau in China by improving photosynthesis rate and water use efficiency (WUE) compared to its native habitat along Yangtze River. Photosynthetic genes were demonstrated as one major category of the candidate genes underlying the physiological superiority. To further study how photosynthetic genes interact to improve the acclimation potential of M. lutarioriparius, population expression patterns within photosynthesis pathway were explored between one mild environment and one harsh environment. We found that 108 transcripts in assembled transcriptome of M. lutarioriparius were highly similar to genes in three Kyoto Encyclopedia of Genes and Genomes (KEGG) photosynthesis pathways of sorghum and maize. Phylogenetic analyses using sorghum, maize, rice, and Arabidopsis genes of dark reaction identified 23 orthologs and 30 paralogs of M. lutarioriparius photosynthetic genes. These genes were also clustered into two kinds of expression pattern. 87% of transcripts in dark reaction were up-regulated and all 14 chloroplast-encoded transcripts in light reaction increased degradation in the harsh environment compared to the mild environment. Moreover, 80.8% of photosynthetic transcripts were coordinated at transcription level under the two environments. Interestingly, LHCI and PSI were significantly correlated with F-ATPase and C4 cycle. Overall, this study indicates the coordinated expression between cyclic electron transport (consisting of LHCI, PSI, and ATPase) and CO2-concentrating mechanism (C4 cycle) could account for photosynthesis plasticity on M. lutarioriparius acclimation potential.

Transcriptome-wide characterization of candidate genes for improving the water use efficiency of energy crops grown on semiarid land.

Understanding the genetic basis of water use efficiency (WUE) and its roles in plant adaptation to a drought environment is essential for the production of second-generation energy crops in water-deficit marginal land. In this study, RNA-Seq and WUE measurements were performed for 78 individuals of Miscanthus lutarioriparius grown in two common gardens, one located in warm and wet Central China near the native habitats of the species and the other located in the semiarid Loess Plateau, the domestication site of the energy crop. The field measurements showed that WUE of M. lutarioriparius in the semiarid location was significantly higher than that in the wet location. A matrix correlation analysis was conducted between gene expression levels and WUE to identify candidate genes involved in the improvement of WUE from the native to the domestication site. A total of 48 candidate genes were identified and assigned to functional categories, including photosynthesis, stomatal regulation, protein metabolism, and abiotic stress responses. Of these genes, nearly 73% were up-regulated in the semiarid site. It was also found that the relatively high expression variation of the WUE-related genes was affected to a larger extent by environment than by genetic variation. The study demonstrates that transcriptome-wide correlation between physiological phenotypes and expression levels offers an effective means for identifying candidate genes involved in the adaptation to environmental changes.

Population transcriptomics reveals a potentially positive role of expression diversity in adaptation.

While it is widely accepted that genetic diversity determines the potential of adaptation, the role that gene expression variation plays in adaptation remains poorly known. Here we show that gene expression diversity could have played a positive role in the adaptation of Miscanthus lutarioriparius. RNA-seq was conducted for 80 individuals of the species, with half planted in the energy crop domestication site and the other half planted in the control site near native habitats. A leaf reference transcriptome consisting of 18,503 high-quality transcripts was obtained using a pipeline developed for de novo assembling with population RNA-seq data. The population structure and genetic diversity of M. lutarioriparius were estimated based on 30,609 genic single nucleotide polymorphisms. Population expression (Ep ) and expression diversity (Ed ) were defined to measure the average level and the magnitude of variation of a gene expression in the population, respectively. It was found that expression diversity increased while genetic diversity decreased after the species was transplanted from the native habitats to the harsh domestication site, especially for genes involved in abiotic stress resistance, histone methylation, and biomass synthesis under water limitation. The increased expression diversity could have enriched phenotypic variation directly subject to selections in the new environment.

Prevalence and clinical characteristics of active epilepsy in southern Han Chinese.

PURPOSE: To investigate the prevalence and clinical characteristics of active epilepsy in southern Han Chinese. METHOD: A door-to-door survey about epilepsy was conducted in communities identified by random cluster sampling among 20 villages and 3 communities of Yueyang city. A questionnaire for epilepsy based on the World Health Organization screening questionnaire was used. A final diagnosis of epilepsy was made by neurology specialists with the support of head magnetic resonance imaging (MRI), computed tomography (CT), and electroencephalography (EEG) if available. The prevalence, clinical characteristics, and treatment gap were analyzed in patients with active epilepsy within the past year and the past 5 years. RESULTS: Active epilepsy was identified in 91 patients within the past year and 117 patients within the past 5 years. The one-year prevalence was 2.8‰, and the five-year prevalence was 3.7‰. The prevalence for epilepsy active within the last year and the last five years was significantly higher in rural areas than in urban areas (P<0.05). Secondary generalized tonic-clonic seizures (53.8%) were the most common seizure type in patients whose epilepsy had been active in the last year. 34.1% of patients were diagnosed with structural or metabolic epilepsy. The most common cause for epilepsy was cerebrovascular disease (32.3%), followed by traumatic brain injury (29.0%). The treatment gap was 93.4%. CONCLUSION: The prevalence of epilepsy active within the last one and five years was higher in rural areas than in urban areas of Yueyang city. A large treatment gap exists in this area and a rational intervention strategy is needed.