A Multi-stage Segmentation Method for Tongue Ecchymosis.

Tongue diagnosis is an important component of traditional Chinese medicine (TCM), in which tongue ecchymosis is the main diagnostic basis for the blood stasis syndrome of TCM. Most of the existing methods are unsupervised and cannot accurately segment tongue ecchymosis. In this paper, we propose a multi-stage segmentation method for tongue ecchymosis. We first employ an object detection model for rough localization of tongue ecchymosis, and then use the unsupervised clustering and the watershed transform for rough segmentation and fine segmentation of tongue ecchymosis respectively. To the best of our knowledge, we are the first to combine machine learning and deep learning to segment tongue ecchymosis. Experimental results show that the tongue ecchymoses obtained by our method are more similar to the real tongue ecchymoses compared with the existing methods, and the Intersection-over-Union (IoU) is improved by 0.12 compared with the latest method.Clinical Relevance-Tongue ecchymosis obtained by this paper is the main diagnostic basis for the blood stasis syndrome of TCM.

Circulating messenger RNA variants as a potential biomarker for surveillance of hepatocellular carcinoma.

Background and rationale: Liver derived messenger ribonucleic acid (mRNA) transcripts were reported to be elevated in the circulation of hepatocellular carcinoma (HCC) patients. We now report the detection of high-risk mRNA variants exclusively in the circulation of HCC patients. Numerous genomic alleles such as single nucleotide polymorphisms (SNPs), nucleotide insertions and deletions (called Indels), splicing variants in many genes, have been associated with elevated risk of cancer. Our findings potentially offer a novel non-invasive platform for HCC surveillance and early detection. Approach: RNAseq analysis was carried out in the plasma of 14 individuals with a diagnosis of HCC, 8 with LC and no HCC, and 6 with no liver disease diagnosis. RNA from 6 matching tumors and 5 circulating extracellular vesicle (EV) samples from 14 of those with HCC was also analyzed. Specimens from two cholangiocarcinoma (CCA) patients were also included in our study. HCC specific SNPs and Indels referred as "variants" were identified using GATK HaplotypeCaller and annotated by SnpEff to filter out high risk variants. Results: The variant calling on all RNA samples enabled the detection of 5.2 million SNPs, 0.91 million insertions and 0.81 million deletions. RNAseq analyses in tumors, normal liver tissue, plasma, and plasma derived EVs led to the detection of 5480 high-risk tumor specific mRNA variants in the circulation of HCC patients. These variants are concurrently detected in tumors and plasma samples or tumors and EVs from HCC patients, but none of these were detected in normal liver, plasma of LC patients or normal healthy individuals. Our results demonstrate selective detection of concordant high-risk HCC-specific mRNA variants in free plasma, plasma derived EVs and tumors of HCC patients. The variants comprise of splicing, frameshift, fusion and single nucleotide alterations and correspond to cancer and tumor metabolism pathways. Detection of these high-risk variants in matching specimens from same subjects with an enrichment in circulating EVs is remarkable. Validation of these HCC selective ctmRNA variants in larger patient cohorts is likely to identify a predictive set of ctmRNA with high diagnostic performance and thus offer a novel non-invasive serology-based biomarker for HCC.

Serum molecular biomarkers in neuromyelitis optica and multiple sclerosis.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is a rare and severe inflammatory demyelinating disorder of the central nervous system (CNS), which mainly affects the optic nerves and spinal cord. The aims of this study were to determine whether the expression levels of serological cytokines could distinguish 1) NMOSD from healthy controls (HCs); and 2) NMOSD patients with and without the aquaporin-4 (AQP4) antibody biomarker from each other; and 3) NMOSD patients without the antibody to AQP4 from MS patients. METHODS: The expression levels of 200 proteins in serum from 41 NMOSD (32 with antibodies to AQP4, 9 without antibodies to AQP4), 12 MS patients, and 34 HCs were measured using glass-based antibody arrays. None of the patients received any immunosuppressive treatment. In parallel, the correlation between protein expression in NMOSD/MS patients and clinical traits was determined with Weighted Gene Co-expression Network Analysis (WGCNA). RESULTS: Thirty-nine serological proteins were differentially expressed in NMOSD patients compared to HCs, with 29 of these proteins not observed in MS patients. In addition, the data reveal 15 differentially-expression proteins (DEPs) between AQP4-IgG seronegative and AQP4-IgG seropositive NMOSD patients, and 9 DEPs between NMOSD and MS patients who did not have AQP4-IgG. CONCLUSION: Serological IL-17B is significantly upregulated in both NMOSD and MS patients compared to HCs, and could be a key biomarker of NMOSD and MS. Serological VEGF, MPIF-1 and NrCAM were positively associated with AQP4-IgG titer. We also demonstrate that EGF may be involved in the breakdown of the BBB by downregulating Claudin-5.

A Case-Control Study of Follicular Fluid Cytokine Profiles in Women with Diminished Ovarian Reserve.

Ovarian reserve is an important determinant of a woman's reproductive potential, and women with diminished ovarian reserve (DOR) often seek in vitro fertilization (IVF). The underlying etiology of DOR is unknown, but follicular fluid cytokine concentrations likely play a role in follicular development and maturation. The present study seeks to investigate the expression of cytokines in follicular fluid (FF) of women with DOR undergoing IVF and explore correlated functional pathways. One hundred ninety-four women undergoing ovarian stimulation were recruited at the time of oocyte retrieval. Women were classified as having DOR if they met one or more of the following criteria: AMH < 1 ng/ml, FSH > 10 mIU/ml, and/or AFC < 10. Controls included women undergoing IVF for male factor, tubal factor due to tubal ligation, or planned oocyte cryopreservation (non-oncologic). The concentrations of 480 cytokines and related growth factors in follicular fluid were determined using a multiplex immunoassay. Fifty-nine cytokines had significantly different concentrations (53 higher and 6 lower) in the DOR relative to the control group after adjusting for age and body mass index (BMI) (false discovery rate; FDR < 0.1). Using the most informative 44 biomarkers as indicated by a random forest (RF) model, an area under the curve (AUC) of 0.78 was obtained. Thus, follicular microenvironment differs between women with DOR and normal ovarian reserve. The differentially expressed cytokines belong to diverse processes that are primarily involved in follicular maturation and ovulation. These changes may play an important role in treatment outcomes in women with DOR.

Statistical Analysis Options for Antibody Array Data.

In this chapter, we introduce the application of R, a statistical programming language in the analysis of antibody array data. We start from a brief introduction of R itself and then cover data filtration and transformation, data visualization, differential expression analysis with/without variance correction, co-expression network, functional enrichment analysis, and statistical modeling.

Next-Generation Glycan Microarray Enabled by DNA-Coded Glycan Library and Next-Generation Sequencing Technology.

Interactions of glycans with proteins, cells, and microorganisms play important roles in cell-cell adhesion and host-pathogen interaction. Glycan microarray technology, in which multiple glycan structures are immobilized on a single glass slide and interrogated with glycan-binding proteins (GBPs), has become an indispensable tool in the study of protein-glycan interactions. Despite its great success, the current format of the glycan microarray requires expensive, specialized instrumentation and labor-intensive assay and image processing procedures, which limit automation and possibilities for high-throughput analyses. Furthermore, the current microarray is not suitable for assaying interaction with intact cells due to their large size compared to the two-dimensional microarray surface. To address these limitations, we developed the next-generation glycan microarray (NGGM) based on artificial DNA coding of glycan structures. In this novel approach, a glycan library is presented as a mixture of glycans and glycoconjugates, each of which is coded with a unique oligonucleotide sequence (code). The glycan mixture is interrogated by GBPs followed by the separation of unbound coded glycans. The DNA sequences that identify individual bound glycans are quantitatively sequenced (decoded) by powerful next-generation sequencing (NGS) technology, and copied numbers of the DNA codes represent relative binding specificities of corresponding glycan structures to GBPs. We demonstrate that NGGM generates glycan-GBP binding data that are consistent with that generated in a slide-based glycan microarray. More importantly, the solution phase binding assay is directly applicable to identifying glycan binding to intact cells, which is often challenging using glass slide-based glycan microarrays.

Predictive model for contrast-enhanced ultrasound of the breast: Is it feasible in malignant risk assessment of breast imaging reporting and data system 4 lesions?

AIM: To build and evaluate predictive models for contrast-enhanced ultrasound (CEUS) of the breast to distinguish between benign and malignant lesions. METHODS: A total of 235 breast imaging reporting and data system (BI-RADS) 4 solid breast lesions were imaged via CEUS before core needle biopsy or surgical resection. CEUS results were analyzed on 10 enhancing patterns to evaluate diagnostic performance of three benign and three malignant CEUS models, with pathological results used as the gold standard. A logistic regression model was developed basing on the CEUS results, and then evaluated with receiver operating curve (ROC). RESULTS: Except in cases of enhanced homogeneity, the rest of the 9 enhancement appearances were statistically significant (P < 0.05). These 9 enhancement patterns were selected in the final step of the logistic regression analysis, with diagnostic sensitivity and specificity of 84.4% and 82.7%, respectively, and the area under the ROC curve of 0.911. Diagnostic sensitivity, specificity, and accuracy of the malignant vs benign CEUS models were 84.38%, 87.77%, 86.38% and 86.46%, 81.29% and 83.40%, respectively. CONCLUSION: The breast CEUS models can predict risk of malignant breast lesions more accurately, decrease false-positive biopsy, and provide accurate BI-RADS classification.

Contrast-enhanced ultrasound improved performance of breast imaging reporting and data system evaluation of critical breast lesions.

AIM: To determine whether contrast-enhanced ultrasound (CEUS) can improve the precision of breast imaging reporting and data system (BI-RADS) categorization. METHODS: A total of 230 patients with 235 solid breast lesions classified as BI-RADS 4 on conventional ultrasound were evaluated. CEUS was performed within one week before core needle biopsy or surgical resection and a revised BI-RADS classification was assigned based on 10 CEUS imaging characteristics. Receiver operating characteristic curve analysis was then conducted to evaluate the diagnostic performance of CEUS-based BI-RADS assignment with pathological examination as reference criteria. RESULTS: The CEUS-based BI-RADS evaluation classified 116/235 (49.36%) lesions into category 3, 20 (8.51%), 13 (5.53%) and 12 (5.11%) lesions into categories 4A, 4B and 4C, respectively, and 74 (31.49%) into category 5. Selecting CEUS-based BI-RADS category 4A as an appropriate cut-off gave sensitivity and specificity values of 85.4% and 87.8%, respectively, for the diagnosis of malignant disease. The cancer-to-biopsy yield was 73.11% with CEUS-based BI-RADS 4A selected as the biopsy threshold compared with 40.85% otherwise, while the biopsy rate was only 42.13% compared with 100% otherwise. Overall, only 4.68% of invasive cancers were misdiagnosed. CONCLUSION: This pilot study suggests that evaluation of BI-RADS 4 breast lesions with CEUS results in reduced biopsy rates and increased cancer-to-biopsy yields.

Phenotypic Heterogeneity in a DFNA20/26 family segregating a novel ACTG1 mutation.

BACKGROUND: Genetic factors play an important role in hearing loss, contributing to approximately 60% of cases of congenital hearing loss. Autosomal dominant deafness accounts for approximately 20% of cases of hereditary hearing loss. Diseases with autosomal dominant inheritance often show pleiotropy, different degrees of penetrance, and variable expressivity. METHODS: A three-generation Chinese family with autosomal dominant nonsyndromic hearing impairment (ADNSHI) was enrolled in this study. Audiometric data and blood samples were collected from the family. In total, 129 known human deafness genes were sequenced using next-generation sequencing (NGS) to identify the responsible gene mutation in the family. Whole Exome Sequencing (WES) was performed to exclude any other variant that cosegregated with the phenotype. RESULTS: The age of onset of the affected family members was the second decade of life. The condition began with high-frequency hearing impairment in all family members excluding III:2. The novel ACTG1 c.638A > G (p.K213R) mutation was found in all affected family members and was not found in the unaffected family members. A heterozygous c.638A > G mutation in ACTG1 and homozygous c.109G > A (p.V37I) mutation in GJB2 were found in III:2, who was born with hearing loss. The WES result concurred with that of targeted sequencing of known deafness genes. CONCLUSIONS: The novel mutation p.K213R in ACTG1 was found to be co-segregated with hearing loss and the genetic cause of ADNSHI in this family. A homozygous mutation associated with recessive inheritance only rarely co-acts with a dominant mutation to result in hearing loss in a dominant family. In such cases, the mutations in the two genes, as in ACTG1 and GJB2 in the present study, may result in a more severe phenotype. Targeted sequencing of known deafness genes is one of the best choices to identify the genetic cause in hereditary hearing loss families.

Combined examination of sequence and copy number variations in human deafness genes improves diagnosis for cases of genetic deafness.

BACKGROUND: Copy number variations (CNVs) are the major type of structural variation in the human genome, and are more common than DNA sequence variations in populations. CNVs are important factors for human genetic and phenotypic diversity. Many CNVs have been associated with either resistance to diseases or identified as the cause of diseases. Currently little is known about the role of CNVs in causing deafness. CNVs are currently not analyzed by conventional genetic analysis methods to study deafness. Here we detected both DNA sequence variations and CNVs affecting 80 genes known to be required for normal hearing. METHODS: Coding regions of the deafness genes were captured by a hybridization-based method and processed through the standard next-generation sequencing (NGS) protocol using the Illumina platform. Samples hybridized together in the same reaction were analyzed to obtain CNVs. A read depth based method was used to measure CNVs at the resolution of a single exon. Results were validated by the quantitative PCR (qPCR) based method. RESULTS: Among 79 sporadic cases clinically diagnosed with sensorineural hearing loss, we identified previously-reported disease-causing sequence mutations in 16 cases. In addition, we identified a total of 97 CNVs (72 CNV gains and 25 CNV losses) in 27 deafness genes. The CNVs included homozygous deletions which may directly give rise to deleterious effects on protein functions known to be essential for hearing, as well as heterozygous deletions and CNV gains compounded with sequence mutations in deafness genes that could potentially harm gene functions. CONCLUSIONS: We studied how CNVs in known deafness genes may result in deafness. Data provided here served as a basis to explain how CNVs disrupt normal functions of deafness genes. These results may significantly expand our understanding about how various types of genetic mutations cause deafness in humans.

Novel compound heterozygous mutations in MYO7A Associated with Usher syndrome 1 in a Chinese family.

Usher syndrome is an autosomal recessive disease characterized by sensorineural hearing loss, age-dependent retinitis pigmentosa (RP), and occasionally vestibular dysfunction. The most severe form is Usher syndrome type 1 (USH1). Mutations in the MYO7A gene are responsible for USH1 and account for 29-55% of USH1 cases. Here, we characterized a Chinese family (no. 7162) with USH1. Combining the targeted capture of 131 known deafness genes, next-generation sequencing, and bioinformatic analysis, we identified two deleterious compound heterozygous mutations in the MYO7A gene: a reported missense mutation c.73G>A (p.G25R) and a novel nonsense mutation c.462C>A (p.C154X). The two compound variants are absent in 219 ethnicity-matched controls, co-segregates with the USH clinical phenotypes, including hearing loss, vestibular dysfunction, and age-dependent penetrance of progressive RP, in family 7162. Therefore, we concluded that the USH1 in this family was caused by compound heterozygous mutations in MYO7A.

[Comparison analysis of multi-modalities images in 158 patients with hepatic tumor].

OBJECTIVE: To compare and analyze the positron emission tomography (PET) images with other multi-modalities in the diagnosis of hepatic tumors. METHODS: A total of 158 patients undergoing (18)F fluorodeoxyglucose (FDG)-PET were enrolled along with another 55 cases with (11)C acetate-PET (AC-PET) imaging within 1 week. The pathological results were taken as the golden criteria. Tumor marker, contrast ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) results were collected.SPSS 19.0 was used for statistical analysis. RESULTS: Contrast MRI, ultrasound and CT were more effective than PET in the differential diagnosis of hepatic tumor.FDG-PET showed a better accuracy in the diagnosis of intrahepatic cholangiocarcinomas, metastasis and rare primary hepatic carcinoma. AC-PET was a good complementary method to FDG-PET. Consideration of tumor size, amount, laboratory results and history could improve the diagnostic accuracy of PET and enhanced CT. CONCLUSION: PET must be combined with other image modalities to differentiate hepatic tumors more effectively and accurately.

Applications of targeted gene capture and next-generation sequencing technologies in studies of human deafness and other genetic disabilities.

The goal of sequencing the entire human genome for $1000 is almost in sight. However, the total costs including DNA sequencing, data management, and analysis to yield a clear data interpretation are unlikely to be lowered significantly any time soon to make studies on a population scale and daily clinical uses feasible. Alternatively, the targeted enrichment of specific groups of disease and biological pathway-focused genes and the capture of up to an entire human exome (~1% of the genome) allowing an unbiased investigation of the complete protein-coding regions in the genome are now routine. Targeted gene capture followed by sequencing with massively parallel next-generation sequencing (NGS) has the advantages of 1) significant cost saving, 2) higher sequencing accuracy because of deeper achievable coverage, 3) a significantly shorter turnaround time, and 4) a more feasible data set for a bioinformatic analysis outcome that is functionally interpretable. Gene capture combined with NGS has allowed a much greater number of samples to be examined than is currently practical with whole-genome sequencing. Such an approach promises to bring a paradigm shift to biomedical research of Mendelian disorders and their clinical diagnoses, ultimately enabling personalized medicine based on one's genetic profile. In this review, we describe major methodologies currently used for gene capture and detection of genetic variations by NGS. We will highlight applications of this technology in studies of genetic disorders and discuss issues pertaining to applications of this powerful technology in genetic screening and the discovery of genes implicated in syndromic and non-syndromic hearing loss.

Melamine-contaminated powdered formula and urolithiasis in young children.

BACKGROUND: A recent epidemic of melamine contamination of baby formula in China has been associated with the development of urinary tract stones, though the clinical manifestations and predisposing factors are incompletely delineated. METHODS: We administered a questionnaire to the parents of children 36 months of age or younger who were being screened for a history of exposure to melamine and symptoms of, and possible predisposing factors for, urinary tract stones. In addition, we performed urinalysis, renal-function and liver-function tests, urinary tests for biochemical markers and the calcium:creatinine ratio, and ultrasonography. Powdered-milk infant formulas were classified as having a high melamine content (>500 ppm), a moderate melamine content (<150 ppm), or no melamine (0 ppm); no formulas contained between 150 and 500 ppm of melamine. RESULTS: Contaminated formula was ingested by 421 of 589 children. Fifty had urinary stones, including 8 who had not received melamine-contaminated formula; 112 were suspected to have stones; and 427 had no stones. Among children with stones, 5.9% had hematuria and 2.9% had leukocyturia, percentages that did not differ significantly from those among children who were suspected to have stones or those who did not have stones. Serum creatinine, urea nitrogen, and alanine aminotransferase levels were normal in the 22 children with stones who were tested. Four of the 41 children (9.8%) who had stones and in whom urinary markers of glomerular function were measured had evidence of abnormalities; none had tubular dysfunction. Children exposed to high-melamine formula were 7.0 times as likely to have stones as those exposed to no-melamine formula. Preterm infants were 4.5 times as likely to have stones as term infants. CONCLUSIONS: Prematurity and exposure to melamine-contaminated formula were associated with urinary stones. Affected children lacked typical signs and symptoms of urolithiasis.

Mortality rate prediction by Physiological and Operative Severity Score for the Enumeration of Mortality and Morbidity (POSSUM), Portsmouth POSSUM and Colorectal POSSUM and the development of new scoring systems in Chinese colorectal cancer patients.

BACKGROUND: The aim of this study was to compare the Physiological and Operative Severity Score for the enumeration of Mortality and Morbidity (POSSUM), Portsmouth POSSUM (P-POSSUM), and Colorectal POSSUM (Cr-POSSUM) for predicting surgical mortality in Chinese colorectal cancer patients and to create new scoring systems to achieve better prediction. METHODS: Data from 903 patients undergoing surgery for colon and rectal cancers from 1992 to 2005 at Peking University Third Hospital were included in this study. POSSUM, P-POSSUM, and Cr-POSSUM were used to predict mortality. Stepwise logistic regression was used to develop the modified P-POSSUM and Cr-POSSUM. Their performances were tested by receiver operating characteristic curve, Hosmer-Lemeshow statistic, and observed:expected ratio. RESULTS: The actual inpatient mortality was 1.0% (9 of 903). The predicted mortality of POSSUM, P-POSSUM, and Cr-POSSUM were 5.6%, 2.8%, and 4.8%, respectively, which were significantly higher than the actual mortality in our cohort. The predicted mortality of the modified P-POSSUM and Cr-POSSUM was very close to the observed mortality. Both the modified models offered better accuracy than P-POSSUM. CONCLUSIONS: The predicted mortality of POSSUM, P-POSSUM, and Cr-POSSUM were significantly higher than the observed mortality in our patients. The modified P-POSSUM and Cr-POSSUM models provided an accurate prediction of inpatient mortality rate in colorectal cancer patients in China.

Intraoperative monitoring for safety of total hip arthroplasty using third-generation cementing technique.

BACKGROUND: Controversies on the safety of the cement application between cemented and uncemented total hip arthroplasty (THA) have been existing for decades. The purpose of this study was to observe the changes in mean arterial pressure (MAP), heart rate (HR) and oxygen pressure (PaO(2)) during cemented THA, and to evaluate the intraoperative safety of using the third-generation cementing technique and investigate whether the intraoperative risk is higher in acute femoral neck fracture patients than non-traumatic patients. METHODS: Forty-two patients who underwent cemented THA between November 2005 and September 2007 were prospectively included in this study. The third-generation cementing technique as vacuum mixing and pulsatile lavage was used strictly. The MAP and HR were monitored and documented during each operation. Blood gas analysis was performed at exposure, cup implantation, stem implantation and wound closure. MAP, HR and PaO(2) were compared between pre- and post-cement application. Comparisons of MAP, HR and PaO(2) between patients with acute femoral neck fracture and non-traumatic patients were performed as well. RESULTS: No intraoperative cardiopulmonary complication occurred in these cases. No obvious changes were observed in MAP, HR and PaO(2) after cement application. There was no significant difference in MAP, HR and PaO(2) between acute femoral fracture patients (18 patients) and non-traumatic patients (24 patients). CONCLUSIONS: The results of this study suggested that the invasive blood pressure monitoring and blood gas analysis are essential for patients undergoing cemented THA, especially for patients with femoral neck fracture. The third-generation cementing technique is safe to use in THA.

Investigation of road-traffic noise and annoyance in Beijing: a cross-sectional study of 4th Ring Road.

The authors aimed to evaluate traffic noise level and noise annoyance in Beijing and the impact of the noise on the quality of life of the residences. The authors performed a cross-sectional study in a 12-floor college dormitory near 4th Ring Road in Beijing, China. The north-side rooms of the building were noisy and had windows facing the road. The authors measured both indoor and outdoor noise. Using both a 5-item verbal scale and a 0-10 numerical scale, they questioned a sample of 1,293 college students living in the dormitory about road-traffic noise annoyance. The results showed that the average outdoor day-to-night noise level was 79.2 dB(A) in the noisy rooms and 64.0 dB(A) in the quiet rooms. Nearly 39% of the respondents living in the noisy rooms indicated that they were highly annoyed by traffic noise according to the response on the verbal scale, and 50% of the respondents living in the noisy rooms were highly annoyed according to the numerical scale.

[Epidemiological investigation on acute respiratory distress syndrome occurring in intensive care units in Beijing from 1998 to 2003].

OBJECTIVE: To investigate incidence, causes and mortality of acute respiratory distress syndrome (ARDS) in intensive care units (ICU) in Beijing. METHODS: Patients in ICU of eight general hospitals in Beijing from May 1998 to April 2003 were retrospectively studied. ARDS was diagnosed according to the 1994 American-European consensus conference criteria. Results Of 8 482 patients admitted to ICUs in the same period, there were 383 patients (4.5%) diagnosed as having ARDS. Major primary diseases for ARDS were sepsis (21.7%), pneumonia (16.2%), surgical operation (13.1%), acute pancreatitis (12.8%) and multiple severe trauma (10.7%). The mean interval between onset of the primary disease and onset of ARDS was (61.8 +/- 43.7) hours. The overall mortality of ARDS was 52.0%, the mortality adjusted for age (< or =39, 40-64, > or =65 years old) and gender (male) showed significant changes in each year (all P<0.05), but acute physiology and chronic health evaluation II (APACHE II, < or =12, 13-19, > or =20 scores) score showed no significant changes during the 5 years. Septic shock (36.2%) and heart failure (20.6%) were major lethal causes, while only 14.6% died of respiratory failure. CONCLUSION: ARDS was frequent in ICU in Beijing, the mortality remains high, and there is no tendency to decline in recent years.