Real-world NUDT15 genotyping and thiopurine treatment optimization in inflammatory bowel disease: a multicenter study.

BACKGROUND: This study evaluated the effectiveness of NUDT15 codon 139 genotyping in optimizing thiopurine treatment for inflammatory bowel disease (IBD) in Japan, using real-world data, and aimed to establish genotype-based treatment strategies. METHODS: A retrospective analysis of 4628 IBD patients who underwent NUDT15 codon 139 genotyping was conducted. This study assessed the purpose of the genotyping test and subsequent prescriptions following the obtained results. Outcomes were compared between the Genotyping group (thiopurine with genotyping test) and Non-genotyping group (thiopurine without genotyping test). Risk factors for adverse events (AEs) were analyzed by genotype and prior genotyping status. RESULTS: Genotyping test for medical purposes showed no significant difference in thiopurine induction rates between Arg/Arg and Arg/Cys genotypes, but nine Arg/Cys patients opted out of thiopurine treatment. In the Genotyping group, Arg/Arg patients received higher initial doses than the Non-genotyping group, while Arg/Cys patients received lower ones (median 25 mg/day). Fewer AEs occurred in the Genotyping group because of their lower incidence in Arg/Cys cases. Starting with < 25 mg/day of AZA reduced AEs in Arg/Cys patients, while Arg/Arg patients had better retention rates when maintaining ≥ 75 mg AZA. Nausea and liver injury correlated with thiopurine formulation but not dosage. pH-dependent mesalamine reduced leukopenia risk in mesalamine users. CONCLUSIONS: NUDT15 codon 139 genotyping effectively reduces thiopurine-induced AEs and improves treatment retention rates in IBD patients after genotype-based dose adjustments. This study provides data-driven treatment strategies based on genotype and identifies risk factors for specific AEs, contributing to a refined thiopurine treatment approach.

Excess heat production of the pair annihilation of ionic vacancies in a copper redox reaction using a double bipolar MHD electrode.

Through a copper double bipolar magnetohydrodynamic (MHD) electrode (MHDE) producing twice the amounts of ionic vacancies than a conventional single MHDE, the molar excess heat of the pair annihilation of ionic vacancies, 702 kJ mol-1 at 10 T on average was obtained in a copper redox reaction. It was about twice as large as that of a single MHDE, 387 kJ mol-1 at the same magnetic field. This result strongly suggests that a multi-channel bipolar MHDE will produce much greater excess heat. To conserve the linear momentum and electric charge during electron transfer in an electrode reaction, ionic vacancies are created, storing the solvation energy in the polarized core of the order of 0.1 nm, and the pair annihilation of the vacancies with opposite charges liberates the energy as excess heat. The promoted excess heat by the double bipolar MHDE with a diffuser at 10 T was 710 ± 144 kJ mol-1, whereas as mentioned above, 702 ± 426 kJ mol-1 was obtained by the same electrode without such a diffuser. From the theoretical excess heat of 1140 kJ mol-1, the collision efficiencies in pair annihilation were 0.623 ± 0.126 and 0.616 ± 0.374, respectively. From these results, the reproducibility of the thermal measurement was experimentally validated. At the same time, it was concluded that at magnetic fields beyond 10 T, the concentration of ionic vacancy and the collision efficiency take constant uppermost values.

Endoscopic Treatment of Colo-Colonic Intussusception in a Patient with Peutz-Jeghers Syndrome.

A 19-year-old man with a history of Peutz-Jeghers syndrome (PJS) and two previous partial small bowel resections because of intussusception presented with lower abdominal pain. Computed tomography (CT) showed concentric multilayer and cord-like structures in the transverse colon. Colo-colonic intussusception was suspected and he was hospitalized. After two therapeutic enemas were unsuccessful, a colonoscopy was performed. The intussusception was reduced and a 40-mm transverse colon polyp with a thick stalk was resected. After the procedure, his abdominal pain was relieved and he was discharged on the sixth hospital day. This case and several previous reports suggest that PJS polyps with tumor diameter exceeding 30 mm and location in the transverse or sigmoid colon can cause intussusception. Endoscopic treatment should be considered for these lesions.

A highly efficient scheme for library preparation from single-stranded DNA.

Although methods for sequencing library preparation from double-stranded DNA are well established, those from single-stranded DNA (ssDNA) have not been well studied. Further, the existing methods have limitations in efficiency and yield. Therefore, we developed a highly efficient procedure for sequencing library preparation from ssDNA. In this method, the first adaptor tagging of ssDNA is performed using terminal deoxyribonucleotidyl transferase (TdT)-assisted adenylate connector-mediated ssDNA (TACS) ligation, which we reported recently. After complementary strand synthesis using the adaptor-tagged ssDNA, second adaptor tagging via Vaccinia virus topoisomerase I (VTopoI or TOPO)-based adaptor ligation is performed. With additional steps for degradation, repression, and removal of the adaptor dimer, the proposed TACS-TOPO scheme realizes adaptor dimer-free sequencing library preparation from ssDNA samples of 24 pg. The TACS-TOPO scheme was successfully applied to cell-free DNA analysis with amplification-free library preparation from 50 µL of human serum. A modified TACS-TOPO scheme was also applied to DNA extracted from ancient human bones, bringing two to eight times more library yields than those using a conventional library preparation protocol. The procedures for preparing VTopoI and its complex with a double-stranded oligonucleotide adaptor are also described. Overall, the proposed TACS-TOPO scheme can facilitate practical and sensitive sequencing analysis of ssDNA.

Post-bisulfite Adaptor Tagging Based on an ssDNA Ligation Technique (tPBAT).

Post-bisulfite adaptor tagging (PBAT) is a concept that enables the preparation of an efficient sequencing library from bisulfite-treated DNA, and it also means the protocol implemented the concept. Although the previous PBAT or rPBAT was sensitive enough for single-cell methylome analysis, the protocol had several drawbacks owing to the repeated random priming reactions. To resolve these problems, we developed a unique single-strand DNA ligation technique, termed TACS ligation, and established a new protocol called tPBAT. With tPBAT, the data quality improved, with a longer insert and higher mapping rate than that obtained with rPBAT. In addition, paired-end sequencing and indexing were supported by the default. In this chapter, the tPBAT protocol is introduced, and a thorough description of its application to small samples is provided.

Identification, expression, and purification of DNA cytosine 5-methyltransferases with short recognition sequences.

BACKGROUND: DNA methyltransferases (MTases) are enzymes that induce methylation, one of the representative epigenetic modifications of DNA, and are also useful tools for analyzing epigenomes. However, regarding DNA cytosine 5-methylation, MTases identified so far have drawbacks in that their recognition sequences overlap with those for intrinsic DNA methylation in mammalian cells and/or that the recognition sequence is too long for fine epigenetic mapping. To identify MTases with short recognition sequences that never overlap with the CG dinucleotide, we systematically investigated the 25 candidate enzymes identified using a database search, which showed high similarity to known cytosine 5-MTases recognizing short sequences. RESULTS: We identified MTases with six new recognition sequences, including TCTG, CC, CNG, TCG, GCY, and GGCA. Because the recognition sequence never overlapped with the CG dinucleotide, MTases recognizing the CC dinucleotide were promising. CONCLUSIONS: In the current study, we established a procedure for producing active CC-methylating MTases and applied it to nucleosome occupancy and methylome sequencing to prove the usefulness of the enzyme for fine epigenetic mapping. MTases that never overlap with CG dinucleotides would allow us to profile multiple epigenomes simultaneously.

Lower effectiveness of intravenous steroid treatment for moderate-to-severe ulcerative colitis in hospitalised patients with older onset: a multicentre cohort study.

BACKGROUND: The increasing incidence of older-onset ulcerative colitis (UC), which has a higher risk of surgery, is a global health issue. However, data regarding intravenous steroid treatment, one of the important treatment options to avoid surgery, for older-onset UC is lacking. AIMS: To evaluate the association between onset age and effectiveness of intravenous steroids in UC. METHODS: This retrospective multicentre (27 facilities) cohort study included moderate-to-severe hospitalised UC patients who underwent their first intravenous steroids between April 2014 and July 2019. The primary outcome was clinical remission at day 30, using two-item patient-reported outcome scoring. The key secondary outcomes were risks of surgery and adverse events (death, infection and venous thrombosis) within 90 days. A modified Poisson regression model was used for analysis. RESULTS: Overall, 467 UC patients (384 younger-onset and 83 older-onset) were enrolled. Clinical remission at day 30 was observed in 252 (65.6%) among younger-onset patients and 43 (51.8%) among older-onset patients (adjusted risk difference, -21.7% [95% CI, -36.1% to -7.2%]; adjusted risk ratio [ARR], 0.74 [95% CI, 0.59 to 0.93]). The risks of surgery and adverse events were higher in older-onset UC (20.5% vs. 3.1%; ARR, 8.92 [95% CI, 4.13 to 19.27], 25.3% vs. 9.1%; ARR, 2.19 [95% CI, 1.22 to 3.92], respectively). Four deaths occurred, all involving older-onset UC. The risks of infection and venous thrombosis were also higher in older-onset UC (18.1% vs. 8.6%, 7.2% vs. 0.5%, respectively). CONCLUSIONS: Older-onset was associated with a lower effectiveness of intravenous steroids with higher risks of surgery and adverse events in UC.

Clinical response of vedolizumab at week 6 predicted endoscopic remission at week 24 in ulcerative colitis.

BACKGROUND AND AIM: Vedolizumab is a humanized monoclonal antibody that selectively inhibits the migration of gut-homing memory T cells into the intestinal submucosa by antagonizing the interaction of α4ß7 integrin with MAdCAM-1. Vedolizumab is employed for ulcerative colitis with moderate to severe activity; however, predictors of its clinical efficacy have not been established in real-world clinical practice. We investigated the clinical characteristics predicting vedolizumab efficacy. METHODS: This was a single-center, retrospective, observational study that enrolled patients with ulcerative colitis at Kyorin University Hospital. Fifty-two consecutive patients who started vedolizumab induction therapy and were tracked for minimum 14 weeks between August 2018 and February 2021 were included. Clinical and endoscopic disease activities were scored at baseline and at weeks 2, 6, and 14 with the Lichtiger index and at baseline and week 24 with the Mayo endoscopic subscore, respectively. Clinical remission, clinical response, and endoscopic remission were defined as Lichtiger index of ≤3, Lichtiger index of ≤10 with a reduction of minimum 3 points from baseline, and Mayo endoscopic subscore of ≤1, respectively. RESULTS: In these cases, clinical response/remission rates at weeks 2, 6, and 14 were 26.9%/15.3%, 50.0%/46.3%, and 57.6%/50.0%, respectively. The endoscopic remission rate at week 24 was 60%. The clinical response at week 6 was significantly associated with endoscopic remission at week 24 after starting vedolizumab. CONCLUSIONS: In vedolizumab treatment for ulcerative colitis, the clinical response at week 6 can be a predictor for endoscopic remission at week 24.

Multicenter, cross-sectional, observational study on Epstein-Barr viral infection status and thiopurine use by age group in patients with inflammatory bowel disease in Japan (EBISU study).

BACKGROUND: The Epstein-Barr virus (EBV) infection status in patients with inflammatory bowel disease (IBD), particularly those using thiopurines, may be associated with the risk of lymphoproliferative disorder and hemophagocytic lymphohistiocytosis. This was the first multicenter survey of EBV infection in Japanese patients with IBD. Factors related to the EBV infection status were also investigated. METHODS: Five tertiary institutions in Japan participated in this study to examine pediatric and adult patients with IBD. Serum EBV anti-viral capsid antigen (VCA) IgG, EBV anti-VCA IgM, and anti-EBV nuclear antigen-antibody were measured in 495 patients with IBD. The patients' information was obtained from their medical records. Prior EBV infection was defined as anti-VCA IgM negativity and anti-VCA IgG positivity (UMIN000033004). RESULTS: The patients' median age was 25 years (range 0-92 years). Of the 495 patients, nine were anti-VCA IgM-positive and 354 were anti-VCA IgG-positive (seroprevalence: 72.8%). The proportion of patients with prior EBV infection was 0% for those aged < 5 years, < 60% for those aged < 30 years, and > 90% for those aged > 30 years. The proportion of EBV-uninfected patients using thiopurines was 28.4% (52/183) for all patients and 51.8% (44/85) for pediatric patients. Age was significantly associated with anti-VCA IgG seropositivity (p < 0.01, odds ratio: 0.902, 95% confidence interval: 0.880-0.925). No cases of lymphoproliferative disorder, hemophagocytic lymphohistiocytosis, or chronic active EBV infection were reported. CONCLUSIONS: Approximately 30% of Japanese patients with IBD were EBV-uninfected, including those using thiopurines. Age was a significant factor for anti-VCA IgG seropositivity.

Linked color imaging versus white light imaging colonoscopy for colorectal adenoma detection: A randomized controlled trial.

BACKGROUND AND AIM: The adenoma detection rate is an important indicator of colonoscopy quality and colorectal cancer incidence. We compared the adenoma detection rates between white light imaging (WLI) and linked color imaging (LCI) colonoscopy. METHODS: Patients undergoing colonoscopy for positive fecal immunochemical tests, follow-up of colon polyps, and abdominal symptoms at three institutions were randomly assigned to the LCI or WLI groups. Mean adenoma number per patient (including based on endoscopists' experience), adenoma detection rate, cecal intubation time, withdrawal time, mean adenoma number per location, and adenoma size were compared. RESULTS: The LCI and WLI groups comprised 494 and 501 patients, respectively. No significant differences in the cecal intubation rate (LCI vs WLI: 99.5% vs 99.4%), cecal intubation time, and withdrawal time were noted between groups. The mean adenoma number per patient was significantly higher in the LCI group than in the WLI group (1.07 vs 0.88, P = 0.04), particularly in the descending [0.12 (58/494) vs 0.07 (35/501), P = 0.01] and sigmoid colon [0.41 (201/494) vs 0.30 (149/501), P ≤ 0.001]. However, the adenoma detection rate was 47.1% in the LCI group and 46.9% in the WLI group, with no significant difference (P = 0.93). The total number of sessile-type adenomas was significantly higher in the LCI group than in the WLI group (346/494 vs 278/501, P = 0.04). As for polyp size, small polyps (≤ 5 mm) were detected at a significantly higher rate in the LCI group (271/494 vs 336/501, P = 0.04). CONCLUSION: Linked color imaging is significantly superior to WLI in terms of mean adenoma number per patient.

Extent of disease affects the usefulness of fecal biomarkers in ulcerative colitis.

BACKGROUND: Fecal biomarkers are considered to be useful surrogate markers for endoscopic activity. Given the mechanisms of fecal biomarkers, we hypothesized that the extent of ulcerative colitis (UC; pancolitis, left-sided colitis, and proctitis) could affect the usefulness of fecal biomarkers for assessing endoscopic and clinical disease activity; however, few studies have evaluated the utility of fecal biomarkers in the disease extent of UC. METHODS: Fecal calprotectin, a fecal immunochemical test for hemoglobin, and fecal lactoferrin were used as fecal biomarkers. UC patients, who underwent colonoscopy within 30 days of the fecal biomarker test, participated in this observational study. Clinical and endoscopic disease activity was assessed using the Lichtiger Index and Mayo endoscopic subscore (MES), respectively. RESULTS: A total of 162 colonoscopies were performed on 133 UC patients. A correlation analysis between each biomarker and the MES for each disease-extent subgroup showed a decreased correlation in the proctitis compared with the other groups. With the exception of proctitis, it was possible to distinguish between MES 0 and MES ≥ 1 with high area-under-the-curve values for fecal calprotectin and fecal lactoferrin. The fecal immunochemical test for hemoglobin was superior at discriminating MES 0 for proctitis. CONCLUSIONS: For the practical application of fecal biomarkers for UC patients, it is necessary to consider disease extent before use. In particular, patients with proctitis exhibit a low correlation between stool biomarkers and endoscopic findings. The usefulness of these biomarkers for endoscopic remission is reduced, except for the fecal immunochemical test for hemoglobin.

Long-Term Electrodeposition under a Uniform Parallel Magnetic Field. 2. Flow-Mode Transition from Laminar MHD Flow to Convection Cells with Two-Dimensional (2D) Nucleation.

Following the analysis of the self-organization of two-dimensional (2D) nuclei in Part 1, the flow-mode transition from laminar magnetohydrodynamics (MHD) flow to convection cells accompanied by 2D nucleation under a uniform parallel magnetic field was theoretically examined using the statistical mechanics of nonequilibrium fluctuation. As a result, it was clarified that secondary nodules of 2D nuclei develop with multiple nucleations during the transition, forming a one-upon-another structure. Then, the evolution of the convection cells as well as the secondary nodules requires unstable growth of the asymmetrical fluctuations by the specific adsorption of an ion. As predicted by the theory, the electrolytic current in copper deposition with specific adsorption of hydrogen ions under a parallel magnetic field developed with time, resulting in a nonlinear steplike curve in a 1200 s deposition time.

Long-Term Electrodeposition under a Uniform Parallel Magnetic Field. 1. Instability of Two-Dimensional Nucleation in an Electric Double Layer.

Under a parallel magnetic field, after long-term copper deposition from an acidic copper sulfate solution, numerous spherical secondary nodules of 10 to 100 µm diameters were formed one upon another in dendritic mode. This is a new type of micro-magnetohydrodynamic (MHD) effect arising from the unstable growth of three-dimensional (3D) and two-dimensional (2D) nuclei by specific adsorption of hydrogen ions (second micro-MHD effect). From the viewpoint of instability in electrodeposition, though 3D nucleation in the diffusion layer is always unstable, with ionic specific adsorption such as hydrogen ions, stable 2D nucleation turns unstable after long-term deposition. The resultant competitive growth of 3D and 2D nuclei produces spherical nodules as their composite, leading to their dendritic growth. Furthermore, though negligibly small, nonequilibrium fluctuations occurring in 2D nucleation migrate with the laminar solution flow caused by Lorentz force (MHD flow). Depending on whether the ionic adsorption is specific or nonspecific, the traveling asymmetrical fluctuation changes the direction to the upstream or downstream side, respectively.

Excess heat production in the redox couple reaction of ferricyanide and ferrocyanide.

In order to establish the universality of the excess heat production in electrochemical reaction, under a high magnetic field, as one of the most fundamental electrochemical reactions, the case of ferricyanide-ferrocyanide redox reaction was examined, where ionic vacancies with ± 1 unit charge were collided by means of magnetohydrodynamic (MHD) flow. As a result, from the pair annihilation of the vacancies with opposite signs, beyond 7 T, excess heat production up to 25 kJ·mol-1 in average at 15 T was observed, which was attributed to the liberation of the solvation energy stored in a pair of the vacancy cores with a 0.32 nm radius, i.e., 112 kJ·mol-1. Difference between the observed and expected energies comes from the small collision efficiency of 0.22 due to small radius of the vacancy core. Ionic vacancy initially created as a by-product of electrode reaction is unstable in solution phase, stabilized by releasing solvation energy. Ionic vacancy utilizes the energy to enlarge the core and stores the energy in it. As a result, solvated ionic vacancy consists of a polarized free space of the enlarged core surrounded by oppositely charged ionic cloud. The accuracy and precision of the measured values were ascertained by in situ standard additive method.

Half-Elemental Diet Shifts the Human Intestinal Bacterial Compositions and Metabolites: A Pilot Study with Healthy Individuals.

METHODS: This prospective study included four healthy volunteers. The subjects continued their dietary habits for 2 weeks after the registration of the study and then started half-ED replacing 900 kcal of the regular diet with ED (time point 1, T1). The subjects continued half-ED for 2 weeks (T2). After the withdrawal of ED, subjects resumed their original dietary habits for 2 weeks (T3). Fecal samples were collected from all subjects at all time points, T1-3. Fecal DNA and metabolites were extracted from the samples. We performed 16S rRNA gene amplicon sequencing and metabolomic analysis to examine the bacterial compositions and intestinal metabolites. RESULTS: There were differences in the gut bacterial compositions and metabolites at each time point as well as overtime changing patterns between subjects. Several bacteria and metabolites including short-chain fatty acids and bile acids altered significantly across the subjects. The bacterial membership and intestinal metabolites at T3 were different from T1 in all subjects. CONCLUSIONS: Half-ED shifts the gut bacterial compositions and metabolites. The changes varied with each individual, while some microbes and metabolites change commonly across individuals. The impact of half-ED may persist even after the withdrawal. This trial is registered with UMIN ID: 000031920.

Two cases in which tofacitinib effectively treated both ulcerative colitis and alopecia areata.

A 40-year-old woman (case 1) visited the hospital complaining of diarrhea and was diagnosed with ulcerative colitis (UC). She was administered 5-aminosalicylic acid (5-ASA), but developed intolerance. Prednisolone (PSL) was administered, and her symptoms improved. However, alopecia areata developed as the PSL was tapered, and her UC relapsed. Adalimumab, Infliximab (IFX), and golimumab were used, but all showed insufficient efficacy. Therefore, we started tofacitinib (TOF). Her bloody stools and diarrhea improved 3 days after TOF administration, and clinical remission occurred on day 14. Her alopecia areata improved 14 days after starting TOF and improved completely during TOF maintenance therapy. A 19-year-old man (case 2) had developed alopecia areata at 10 years old and was diagnosed with UC at 17 years old. He achieved sustained remission with IFX, but then stopped IFX to receive a live vaccination. His UC relapsed 4 months later, immediately after the live vaccine was administered. Vedolizumab was administered, but was ineffective, as was re-administration of IFX. TOF was administered, and his clinical symptoms improved 7 days later. He achieved clinical remission on day 20. In addition, his hair began to regrow 14 days after starting TOF.

Elevated fecal calprotectin and lactoferrin associated with small intestinal lesions in patients with Behçet disease.

BACKGROUND AND AIMS: Small intestinal lesions in patients with Behçet disease (BD) have a risk of perforation and hemorrhage requiring surgery. However, no screening strategy for such lesions has been established. We investigated small intestinal lesions in BD patients with video capsule endoscopy (VCE) and analyzed clinical characteristics to identify noninvasive biomarkers of such lesions. METHODS: This study included 33 BD patients who underwent VCE (PillCam® SB3) at our institution from June 2016 to January 2019. Clinical characteristics, including age, sex, disease duration, body mass index, gastrointestinal symptoms, eye involvement, and blood examinations, were obtained from the medical records of 27 of the 33 patients. Fecal immunochemical tests for hemoglobin, fecal calprotectin (FC), and fecal lactoferrin (FL) were measured. VCE findings of 145 healthy Japanese individuals from a previous report were used as controls. RESULTS: Two intestinal BD patients were included in the 27 patients. We observed that BD patients exhibit more small intestinal lesions compared with healthy individuals, including erosions, ulcers, and total lesions (erosions or ulcers). FC and FL levels were significantly higher in patients with versus without small intestinal lesions (P = 0.034 and P = 0.046, respectively). Receiver operating characteristic analyses demonstrated that FC (cutoff value = 119 µg/g) and FL (cutoff value = 17 µg/g) were biomarkers for small intestinal lesions in patients with BD. CONCLUSION: The present study using VCE showed that patients with BD had more small intestinal lesions than healthy individuals. FC and FL could be useful for screening BD patients who may have small intestinal lesions.

MEFV Gene-Related Enterocolitis Account for Some Cases Diagnosed as Inflammatory Bowel Disease Unclassified.

BACKGROUND AND AIMS: Familial mediterranean fever (FMF), an autoinflammatory disease, is characterized by periodic fever and serositis. An MEFV gene mutation has been identified as the cause of FMF. Recently, patients with MEFV gene mutations and chronic gastrointestinal mucosal inflammation mimicking inflammatory bowel disease (IBD) have been reported. In this retrospective study, we analyzed the clinical characteristics of patients with IBD unclassified (IBDU) with MEFV gene mutations. METHODS: MEFV gene analysis was performed on 8 patients with IBDU among 710 patients with IBD who had been treated at Kyorin University Hospital from April 2016 to December 2018. Clinical manifestations, endoscopic findings, and serological markers were also analyzed. RESULTS: The average of the 8 patients with IBDU (3 men, 5 women) was 32.7 ± 6.4 years (range 26-76 years). Their symptoms comprised diarrhea (n = 8, 100%), hematochezia (n = 3, 37.5%), abdominal pain (n = 3, 37.5%), high fever (n = 2, 16.5%), and other periodic symptoms (n = 2, 16.5%). MEFV gene mutation was confirmed in 4/8 of these patients. Colonoscopy showed various mucosal lesions, rectal sparing, right side dominant colitis, pseudopolyposis, and granular protrusions. Colchicine was administered to 5 of the 8 patients (4 with and 1 without MEFV mutation) who were resistant to conventional treatment for ulcerative colitis. Clinical and endoscopic improvement was observed in all of 5 patients treated with colchicine. CONCLUSIONS: Some patients diagnosed as having IBDU have enterocolitis related to MEFV gene mutation and respond to colchicine therapy.