A highly sensitive reporter system to monitor endogenous YAP1/TAZ activity and its application in various human cells.

The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity in vitro and Venus fluorescence in vivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/ß-catenin/TCF- and IL-1ß/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and ß-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts.

RNA sequencing and target long-read sequencing reveal an intronic transposon insertion causing aberrant splicing.

More than half of cases with suspected genetic disorders remain unsolved by genetic analysis using short-read sequencing such as exome sequencing (ES) and genome sequencing (GS). RNA sequencing (RNA-seq) and long-read sequencing (LRS) are useful for interpretation of candidate variants and detection of structural variants containing repeat sequences, respectively. Recently, adaptive sampling on nanopore sequencers enables target LRS more easily. Here, we present a Japanese girl with premature chromatid separation (PCS)/mosaic variegated aneuploidy (MVA) syndrome. ES detected a known pathogenic maternal heterozygous variant (c.1402-5A>G) in intron 10 of BUB1B (NM_001211.6), a known responsive gene for PCS/MVA syndrome with autosomal recessive inheritance. Minigene splicing assay revealed that almost all transcripts from the c.1402-5G allele have mis-splicing with 4-bp insertion. GS could not detect another pathogenic variant, while RNA-seq revealed abnormal reads in intron 2. To extensively explore variants in intron 2, we performed adaptive sampling and identified a paternal 3.0 kb insertion. Consensus sequence of 16 reads spanning the insertion showed that the insertion consists of Alu and SVA elements. Realignment of RNA-seq reads to the new reference sequence containing the insertion revealed that 16 reads have 5' splice site within the insertion and 3' splice site at exon 3, demonstrating causal relationship between the insertion and aberrant splicing. In addition, immunoblotting showed severely diminished BUB1B protein level in patient derived cells. These data suggest that detection of transcriptomic abnormalities by RNA-seq can be a clue for identifying pathogenic variants, and determination of insert sequences is one of merits of LRS.

Age dependent discrepancy between SARS-CoV-2 anti-nucleoprotein antibody and anti-RBD spike protein antibody in children reflects vaccine coverage.

BACKGROUND: COVID-19 has become widespread in Japanese children. However, the impact of varying immunization coverage on the seroprevalence of SARS-CoV-2 in children is unknown. METHODS: We examined the SARS-CoV-2 antibody in children aged 0 to 18 who were hospitalized at a university hospital from June 2020 through May 2023. The SARS-CoV-2 anti-nucleoprotein (N) antibody and anti-RBD spike (S) protein antibody was measured. RESULTS: A total of 586 cases were enrolled. The median age was 4 years old (interquartile range 1-9), and 362 (61.8 %) were male. The seroprevalence of anti-S antibodies gradually increased from October 2021 and reached 60 percent by early 2023. The anti-N antibody increased starting in January 2022 and reached 50 percent in May 2023. There was a discrepancy in the seroprevalence of anti-S and N antibodies in children 0 years of age or 12 years and older until the fall of 2022. This discrepancy was minimal for children 1-4 years of age and relatively small in the 5-11-year-old group. DISCUSSION: The data suggests that approximately half of the children in our cohort had been infected with SARS-CoV-2 by May 2023. The discrepancy in seropositivity between the anti-S and N antibodies corresponded to the reported vaccine uptake of each target age group, which suggested protective effects of immunization. However, this effect appeared to diminish after early 2023. CONCLUSION: Age dependent discrepancy between SARS-CoV-2 anti-N and anti-S antibody in children reflected differences in vaccine coverage.

Detailed tracking of antigen and antibody levels during coronavirus disease 2019 treatment in an immunosuppressed patient with anti-neutrophil cytoplasmic autoantibody-associated vasculitis.

A 67-year-old woman with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis was not vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was on multiple immunosuppressive drugs. She was hospitalized because of interstitial shadowing in the lungs and diagnosed with persistent coronavirus disease 2019 (COVID-19). Despite treatment with a recombinant monoclonal antibody and antivirals, her symptoms persisted and she lacked a specific antibody response. She tested negative for SARS-CoV-2 antigen after the second antiviral treatment, and a subsequent chest radiograph showed improvement. However, the antibody levels did not change. This case highlights the importance of careful monitoring of the SARS-CoV-2 antigen and antibody levels during COVID-19 treatment in patients with immunosuppression.

Cycle Threshold (Ct) Values of SARS-CoV-2 Detected with the GeneXpert® System and a Mutation Associated with Different Target Gene Failure.

SARS-CoV-2 nucleic acid detection tests enable rapid virus detection; however, it is challenging to identify genotypes to comprehend the local epidemiology and infection routes in real-time qRT-PCR. At the end of June 2022, our hospital experienced an in-hospital cluster of COVID-19. When examined using the GeneXpert® System, the cycle threshold (Ct) value of the N2 region of the nucleocapsid gene of SARS-CoV-2 was approximately 10 cycles higher than that of the envelope gene. Sanger sequencing revealed a G29179T mutation in the primer and probe binding sites. A review of past test results revealed differences in Ct values in 21 of 345 SARS-CoV-2-positive patients, of which 17 cases were cluster-related and 4 were not. Including these 21 cases, 36 cases in total were selected for whole-genome sequencing (WGS). The viral genomes in the cluster-related cases were identified as BA.2.10, and those in the non-cluster cases were closely related and classified as being downstream of BA.2.10 and other lineages. Although WGS can provide comprehensive information, its use is limited in various laboratory settings. A measurement platform reporting and comparing Ct values of different target genes can improve test accuracy, enhance our understanding of infection spread, and be applied to the quality control of reagents.

Association of functional (GA)n microsatellite polymorphism in the FLI1 gene with susceptibility to human systemic sclerosis.

OBJECTIVE: Susceptibility genes that can account for characteristic features of SSc such as fibrosis, vasculopathy and autoimmunity remain to be determined. In mice, deficiency of Friend leukaemia integration 1 transcription factor (Fli1) causes SSc-like disease with these features. The human FLI1 gene contains (GA)n microsatellite, which has been shown to be associated with expression level. Because microsatellite polymorphisms are difficult to capture by genome-wide association studies, we directly genotyped FLI1 (GA)n microsatellite and examined its association with SSc. METHODS: Genomic DNA from 639 Japanese SSc patients and 851 healthy controls was genotyped for (GA)n microsatellite using the fragment assay. The cut-off repeat number for susceptibility to SSc was determined by receiver operating characteristics (ROC) analysis. Association with susceptibility and clinical characteristics was examined using logistic regression analysis. FLI1 mRNA levels were determined using quantitative RT-PCR. RESULTS: Based on the ROC analysis, (GA)n alleles with ≥22 repeats were collectively defined as L alleles and alleles with ≤21 repeats as S alleles. (GA)n L alleles were significantly associated with susceptibility to SSc (P = 5.0e-04, odds ratio 1.34, additive model). Significant association was observed both in diffuse cutaneous and limited cutaneous SSc. Among the SSc, (GA)n L alleles were significantly enriched in the patients with a modified Rodnan total skin thickness score ≥10 compared with those with a score <10. FLI1 mRNA levels were significantly decreased in healthy controls carrying (GA)n L alleles as compared with non-carriers. CONCLUSION: Extended repeat alleles of FLI1 (GA)n microsatellite may be associated with lower FLI1 mRNA levels and susceptibility to human SSc.

Association of ETS1 polymorphism with granulomatosis with polyangiitis and proteinase 3-anti-neutrophil cytoplasmic antibody positive vasculitis in a Japanese population.

ETS proto-oncogene 1, transcription factor (ETS1) is involved in various immune responses. Genome-wide association studies on systemic lupus erythematosus in Chinese populations identified the association of ETS1 polymorphism in 3' untranslated region, rs1128334A, which was associated with lower ETS1 expression. In view of substantial sharing of susceptibility genes across multiple autoimmune diseases, we examined whether ETS1 is associated with a rare autoimmune rheumatic disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Association of rs1128334 was tested in 466 Japanese patients with AAV and 1099 healthy controls by logistic regression analysis under the additive model. AAV patients were classified into 285 microscopic polyangiitis (MPA), 92 granulomatosis with polyangiitis (GPA), 56 eosinophilic GPA, and 33 unclassifiable AAV, according to the European Medicines Agency (EMEA) algorithm. Among the patients, 376 were positive for MPO-ANCA and 62 for PR3-ANCA. When the patients were classified according to the EMEA classification, rs1128334A allele was significantly increased in GPA (P = 0.0060, P c = 0.030, odds ratio (OR), 1.54; 95% confidence interval (CI), 1.13-2.10). With respect to the ANCA specificity, significant association was observed in PR3-ANCA positive AAV (P = 0.0042, P c = 0.021, OR, 1.72; 95% CI, 1.19-2.49). In conclusion, ETS1 polymorphism was suggested to be associated with GPA and PR3-ANCA positive AAV in a Japanese population.

Gene regulation of two ferredoxin:NADP+ oxidoreductases by the redox-responsive regulator SurR in Thermococcus kodakarensis.

The redox-responsive regulator SurR in the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus kodakarensis binds to the SurR-binding consensus sequence (SBS) by responding to the presence of elemental sulfur. Here we constructed a surR gene disruption strain (DTS) in T. kodakarensis, and identified the genes that were under SurR control by comparing the transcriptomes of DTS and parent strains. Among these genes, transcript levels of ferredoxin:NADP+ oxidoreductases 1 and 2 (FNOR1 and FNOR2) genes displayed opposite responses to surR deletion, indicating that SurR repressed FNOR1 transcription while enhancing FNOR2 transcription. Each promoter region contains an SBS upstream (uSBS) and downstream (dSBS) of TATA. In addition to in vitro binding assays, we examined the roles of each SBS in vivo. In FNOR1, mutations in either one of the SBSs resulted in a complete loss of repression, indicating that the presence of both SBSs was essential for repression. In FNOR2, uSBS indeed functioned to enhance gene expression, whereas dSBS functioned in gene repression. SurR bound to uSBS2 of FNOR2 more efficiently than to dSBS2 in vitro, which may explain why SurR overall enhances FNOR2 transcription. Further analyses indicated the importance in the distance between uSBS and TATA for transcriptional activation in FNOR2.

Prognostic Role of Interferon-λ3 in Anti-Melanoma Differentiation-Associated Gene 5-Positive Dermatomyositis-Associated Interstitial Lung Disease.

OBJECTIVE: Interferon-λ3 (IFNλ3) is a cytokine with antiviral functions on barrier surfaces, and it is associated with disease activity in autoimmune diseases. This study assessed the clinical significance of serum IFNλ3 levels in polymyositis/dermatomyositis (PM/DM)-associated interstitial lung disease (ILD). METHODS: We measured serum IFNλ3 levels in 221 patients with PM/DM-ILD (155 in the derivation cohort, 66 in the validation cohort) and 38 controls. We evaluated factors associated with mortality risk among 79 patients with anti-melanoma differentiation-associated gene 5 (MDA5) antibody-positive DM-ILD. RESULTS: Serum IFNλ3 levels at diagnosis were significantly higher in patients with PM/DM-ILD than in healthy controls. Remarkably, serum IFNλ3 levels were specifically increased in patients with anti-MDA5 antibody-positive DM-ILD in both the derivation and validation cohorts. In anti-MDA5 antibody-positive DM-ILD, patients with high IFNλ3 levels (>120 pg/mL) had significantly lower survival rates than those with low IFNλ3 levels (≤120 pg/mL). A multivariate analysis revealed that high IFNλ3 levels, as well as old age and low Pao2, were significantly associated with poor prognoses in patients with anti-MDA5 antibody-positive DM-ILD. In a classification analysis of patients with anti-MDA5 antibody-positive DM-ILD based on age, IFNλ3 level, and Pao2, patients with old age (>53 years), high IFNλ3 levels (>120 pg/mL), and low Pao2 (<75 mm Hg) had the worst survival. In lung pathologic analyses, IFNλ3-positive staining was observed in macrophages, airway epithelial cells, the pleural region, and intrapulmonary veins in patients with anti-MDA5 antibody-positive DM-ILD. CONCLUSION: Serum IFNλ3 is a promising biomarker for identifying patients at high risk of poor outcomes in anti-MDA5 antibody-positive DM-ILD.

Impact of intensive infection control team activities on the acquisition of methicillin-resistant Staphylococcus aureus, drug-resistant Pseudomonas aeruginosa and the incidence of Clostridium difficile-associated disease.

The transmission of multidrug-resistant organisms (MDROs) is an emerging problem in acute healthcare facilities. To reduce this transmission, we introduced intensive infection control team (ICT) activities and investigated the impact of their introduction. This study was conducted at a single teaching hospital from 1 April 2010 to 31 March 2012. During the intervention period, all carbapenem use was monitored by the ICT, and doctors using carbapenems inappropriately were individually instructed. Information related to patients with newly identified MDROs was provided daily to the ICT and instructions on the appropriate infection control measures for MDROs were given immediately with continuous monitoring. The medical records of newly hospitalized patients were reviewed daily to check previous microbiological results and infection control intervention by the ICT was also performed for patients with a previous history of MDROs. Compared with the pre-intervention period, the antimicrobial usage density of carbapenems decreased significantly (28.5 vs. 17.8 defined daily doses/1000 inpatient days; p < 0.001) and the frequency of use of sanitary items, especially the use of aprons, increased significantly (710 vs 1854 pieces/1000 inpatient days; p < 0.001). The number of cases with hospital-acquired MRSA (0.66 vs. 0.29 cases/1000 inpatient days; p < 0.001), hospital-acquired drug-resistant Pseudomonas aeruginosa (0.23 vs. 0.06 cases/1000 inpatient days; p = 0.006) and nosocomial Clostridium difficile-associated disease (0.47 vs. 0.11 cases/1000 inpatient days; p < 0.001) decreased significantly during the intervention period. Our study showed that proactive and continuous ICT interventions were effective for reduction of MDRO transmission.

Utility of the MBT STAR-Cepha kit in detecting extended-spectrum ß-lactamase producers in clinical urine samples and positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Urinary tract infection (UTI) and bloodstream infection (BSI) caused by extended-spectrum ß-lactamase (ESBL)-producing bacteria are important healthcare problems. Appropriate infection management requires the direct detection of organisms in clinical specimens. We assessed the capability of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based MBT STAR-Cepha kit to detect ESBL producers in clinical urine and blood samples. Over a 1-year period, 90 urine samples and 55 positive monomicrobial (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, or Proteus mirabilis) blood cultures were collected from patients with UTI or BSI at Hamamatsu University Hospital. ß-lactamase activity in these samples was directly detected using the MBT STAR-Cepha kit, and the results were compared with those of antimicrobial susceptibility testing and polymerase chain reaction detection assay for the isolates. In receiver operating characteristic curve analysis, the kit assay showed low accuracy in detecting ESBL producers in urine samples (area under the curve [AUC], 0.69). Meanwhile, the AUC for detecting all ESBL-producing bacteria in positive blood cultures was 0.81. The kit assay detected cefotaxime (CTX) resistance (mainly in CTX-M-type ESBL producers) with high accuracy in positive blood cultures; however, it did not accurately detect ESBL producers in urine samples and CTX-susceptible isolates with other ESBL-associated genes (e.g., TEM and SHV types) in positive blood cultures. MBT STAR-Cepha testing can accurately discriminate CTX-resistant ESBL producers in BSI cases and thus can contribute to effective infection management. The results suggest that different sample types, antibiotic resistance profiles, and resistance genes can affect the kit performance.

Analytical Performance of a Novel Latex Turbidimetric Immunoassay, "Nanopia TARC", for TARC/CCL17 Measurement: A Retrospective Observational Study.

Thymus- and activation-regulated chemokine (TARC, also known as CCL17) is used as a biomarker for atopic dermatitis. The methods currently used for its measurement are complex, time-consuming, and require large machinery, warranting the need for a method that is simple, has a quick turnaround time, and requires less complex machinery. We evaluated the analytical performance of a novel latex turbidimetric immunoassay method, "Nanopia TARC", on 174 residual serum samples from patients with skin or allergic diseases. This evaluation included the assessment of the limit of blank/detection/quantification (LOB/D/Q), precision, accuracy, linearity, interference, and commutability between Nanopia TARC and "HISCL TARC", based on the chemiluminescent enzyme immunoassay (CLEIA) method. The LOB/D/Q values were 13, 57, and 141 pg/mL, respectively. The coefficient of variation of the repeatability was 0.9-3.8%, and that of the intermediate precision was 2.1-5.4%. The total error of the accuracy was 1.9-13.4%. The linearity was 141 and 19,804 pg/mL for TARC. The correlation coefficient between Nanopia TARC and HISCL TARC determined using the Passing-Bablok regression analysis was 0.999. Furthermore, the concordance of diagnostic criteria with AD was 92%. Nanopia TARC was confirmed to have the same analytical performance for TARC measurement as the existing CLEIA method.

Differential Dynamics of Humoral and Cell-Mediated Immunity with Three Doses of BNT162b2 SARS-CoV-2 Vaccine in Healthcare Workers in Japan: A Prospective Cohort Study.

Vaccines against SARS-CoV-2 with good efficacy are now available worldwide. However, gained immunity diminishes over time. Here, we investigate the course of both humoral and cell-mediated immunity in response to three doses of the Pfizer mRNA BNT162b2 SARS-CoV-2 vaccine in healthcare workers in Japan. SARS-CoV-2 anti-receptor-binding domain (RBD) antibodies (total Ig, IgG), neutralizing antibodies (NAb), and ELISpot were measured in serum and whole blood samples collected after each vaccine dose. ELISpot numbers were higher than the cutoff values in most participants at all times. It was suggested that the difference in behavior between humoral immunity and cell-mediated immunity with age is complementary. Anti-RBD total Ig, IgG, and NAb indicated a high correlation at each time point after vaccine doses. Total Ig was retained long-term after the second dose and increased significantly faster by the booster dose than IgG. Nab levels of all subjects were ≤20% six months after the second dose, and the correlation coefficient was greatly reduced. These are due to the avidity of each antibody and differences among commercial kits, which may affect the evaluation of immunokinetics in previous COVID-19 studies. Therefore, it is necessary to harmonize reagents categorized by the same characteristics.

Highly Sensitive and Quantitative Diagnosis of SARS-CoV-2 Using a Gold/Platinum Particle-Based Lateral Flow Assay and a Desktop Scanning Electron Microscope.

The gold standard test for identifying SARS-CoV-2, the causative agent of COVID-19, is polymerase chain reaction (PCR). Despite their limited sensitivity, SARS-CoV-2 antigen rapid diagnostic tests are vital tools in the fight against viral spread. Owing to its simplicity and low cost, the lateral flow assay (LFA) is the most extensively used point-of-care diagnostic test. Here, we report a newly designed LFA-NanoSuit method (LNSM) that works in conjunction with desktop scanning electron microscopy (SEM) to detect SARS-CoV-2. LNSM requires no standard SEM treatment, avoids cellulose and residual buffer deformation, and enables the capture of high-resolution images of antibody-labeled gold/platinum particles reacting with SARS-CoV-2 antigens. To assess its applicability, we compared clinical SARS-CoV-2 samples via visual detection of LFA, LSNM detection of LFA, and real-time reverse transcription-PCR (qRT-PCR). Compared to qRT-PCR, LNSM showed 86.7% sensitivity (26/30; 95% confidence interval (CI): 69.28-96.24%) and 93.3% specificity (14/15; 95% CI: 68.05-99.83%) for SARS-CoV-2. In samples with a relatively low SARS-CoV-2 RNA copy number (30 < Ct ≤ 40), the sensitivity of LNSM was greater (73.3%) than that of visual detection (0%). A simple, sensitive, and quantitative LNSM can be used to diagnose SARS-CoV-2.

Synthetic and natural MOR zeolites as high-capacity adsorbents for the removal of nitrous oxide.

N2O is typically present as a trace gas in chemical processes, but its emission causes serious environmental issues. We herein demonstrate that ion-exchanged mordenite zeolites (framework code: MOR) can exhibit high capacities for N2O adsorption under ambient conditions. In particular, a natural MOR zeolite gives an adsorption capacity as high as 0.34 mmol-N2O per g-zeolite (1 atm, 25 °C), representing the best performing material among all zeolite-based adsorbents reported so far. The results contribute toward a comprehensive understanding of the structure-activity relationship and offer insights to establishing a zeolite-based adsorption system for enriching or removing N2O.

Effect of layered application on the skin permeation of a cosmetic active component, rhododendrol.

Cosmetics containing rhododendrol (RD) were voluntarily recalled after incidents of leukoderma related to their use. Users reported using up to five different RD-containing products by layered application. In this study, we investigated the effects of layered application, formulations, and their components on the skin permeation of cosmetics containing RD. Experiments were designed to simulate actual in-use conditions, such as varying application volumes, physical mixing of formulations, sequence of cosmetics application and time interval between applications, to establish their effect on the skin permeation of RD. Milk and lotion RD-containing cosmetics (2%), 1% aqueous RD, and preparations of formulation components were applied as the first or second layers as finite doses of 10 or 20 µL/cm2. Permeation experiments were performed through excised porcine ear skin using Franz diffusion cells with an effective diffusion area of 1.77 cm2. Cosmetics applied by layered application exhibited lower skin permeation of RD compared with a single application despite having the same application dose. High initial volume (20 µL at 0 or 5 sec) did not exhibit any significant reduction in the permeation of RD. Formulations and their components caused varying reductions in RD permeation, probably due to changes in thermodynamic activity of the active component. Layered application, formulation components, application volume, time interval and sequence of application had significant influences on the skin permeation of the active component. Moreover, this study established a method of investigating the influence of formulations and their components on the skin permeation of actives after layered application.

Thiamine Concentrations in Newly Hospitalized Elderly Patients with Infectious Diseases at a Community Hospital in Japan.

The association between advanced age and the thiamine concentration has not been conclusively determined. A recent report from Japan showed that more than half of nursing home elderly residents at an institution had a low whole-blood thiamine concentration (<20 ng/mL). Therefore, a high incidence of low thiamine concentrations among hospitalized elderly has been anticipated in the Japanese population but never investigated. We evaluated the whole thiamine concentration in newly hospitalized elderly patients (≥65 y old) with infectious diseases. Evaluations were performed on admission and at days 6-8 of hospitalization with liquid chromatography tandem mass spectrometry (LC/MS/MS). As a result, we enrolled a total of 471 patients from September 2015 to December 2016. The median thiamine concentration was 46 ng/mL (IQR, 37-58 ng/mL). Only 7 patients (1%) had thiamine concentrations below 20 ng/mL (66 nmol/L) on admission. Five of these patients were bedridden and unable to eat food by themselves, and the other two patients used loop diuretics for chronic heart failure. The thiamine concentration declined in most patients (84%) at days 6-8 of admission, regardless of their dietary intake during hospitalization. In conclusion, a low thiamine concentration was not prevalent among newly hospitalized elderly patients with infectious diseases. However, the thiamine concentration significantly decreased during the 6-8 d of hospitalization.