Overview of the 87th Annual Scientific Meeting of the Japanese Circulation Society (JCS2023)　- New Challenge With Next Generation.

The 87thAnnual Meeting of the Japanese Circulation Society (JCS2023) was held in March 2023 in Fukuoka, Japan, marking the first in-person gathering after the COVID-19 pandemic. With the theme of "New Challenge With Next Generation" the conference emphasized the development of future cardiovascular leaders and technologies such as artificial intelligence (AI). Notable sessions included the Mikamo Lecture on heart failure and the Mashimo Lecture on AI in medicine. Various hands-on sessions and participatory events were well received, promoting learning and networking. Post-event surveys showed high satisfaction among participants, with positive feedback on face-to-face interactions and the overall experience. JCS2023, attended by 17,852 participants, concluded successfully, marking a significant milestone in post-pandemic meetings, and advancing cardiovascular medicine.

[Collision Tumor Involving Primary Ascending Colon Cancer and Malignant Lymphoma].

A 78-year-old male was diagnosed with a primary gastric B-cell malignant lymphoma and metastatic lung tumor 10 years ago. He underwent chemotherapy at another hospital, achieved complete remission, and was actively undergoing follow- up. He presented to our hospital with a 1-month history of a bulge in his right lower abdomen. CT revealed thickening of the ascending colon and dilatation of the oral intestine. He was diagnosed with ascending colon cancer and underwent right hemicolectomy. The subsequent pathological examination revealed a collision tumor involving diffuse, large B-cell lymphoma and well-differentiated adenocarcinoma. He was discharged from our hospital and received chemotherapy at another institution. Unfortunately, the patient died of interstitial pneumonia 31 months postoperatively. This report describes the resection of a collision tumor involving ascending colon cancer and malignant lymphoma. Surgical treatment combined with postoperative chemotherapy improved this patient's long-term survival.

[Laparoscopic Tumor Resection for Asymptomatic Paraganglioma-A Case Study].

A 55-year-old man was referred for the close examination of an abdominal mass noted on abdominal ultrasonography during a physical examination. A contrast-enhanced computed tomography(CT)scan of his abdomen revealed a 36-mm heterogeneously contrast-enhanced mass on the left side of the aorta. He was referred for laparoscopic tumor resection without preoperative histological examination. The tumor was identified from the dorsal aspect of the mesentery of the transverse colon and was resected only because it was detachable from the duodenum. A temporary abnormal hypertension was observed intraoperatively. However, he exhibited a favorable postoperative course and was discharged on the 8th postoperative day. Grossly, it was a nodular tumor with a diameter of 38 mm. Histopathological examination revealed that the tumor cells having abundant cytoplasm formed large foci and were surrounded by sinusoidal vessels. Immunohistochemistry results were positive for chromogranin A, synaptophysin, and neural cell adhesion molecule; thus, paraganglioma was diagnosed. Herein, we report a case of laparoscopic resection of an asymptomatic paraganglioma.

[Malignant Melanoma of a Male Breast That Was Difficult to Differentiate from Breast Cancer-A Case Report].

INTRODUCTION: Malignant melanoma in the male breast is extremely rare. Here we report a case of malignant melanoma in which a small cystic lesion in the male breast gradually increased during follow-up and was difficult to distinguish from breast cancer. CASE: A 65-year-old male was diagnosed with a tumor in the right breast and was referred to our department for further examination. At 42 years of age, he underwent tumor resection of a malignant melanoma of the abdominal skin. Mammary ultrasonography showed a 0.6 cm cystic mass in his right breast. Eight months later, the right breast mass had increased to 1.4 cm, and a core needle biopsy suggested breast cancer. Total mastectomy with axillary lymph node dissection was performed. HE staining of the resected tumor showed intranuclear inclusion bodies and some large nucleoli. On the basis of various immunostaining methods, malignant melanoma was diagnosed instead of breast cancer. After surgery, adjuvant chemotherapy with molecularly targeted drugs was administered. DISCUSSION: This might have been a case of male breast metastasis of malignant melanoma with very late recurrence.

TCR function analysis using a novel system reveals the multiple unconventional tumor-reactive T cells in human breast cancer-infiltrating lymphocytes.

Tumor-infiltrating lymphocytes (TILs) are a potent source for obtaining tumor-reactive T cell receptors (TCRs). Although comprehensive methods to analyze the TCR repertoire in TILs have been reported, the evaluation system for TCR-reactivity to endogenously expressed antigen in tumor cells remains laborious and time consuming. Consequently, very limited numbers of TCRs in TILs have been analyzed for their reactivity to tumor cells. In this study, we developed an efficient evaluation system for TCR function designated c-FIT (comprehensive functional investigation of TCRs) to analyze TCR reactivity. The c-FIT system enabled us to analyze up to 90 TCRs for their reactivity to tumor cells by a single assay within a month. Using c-FIT, we analyzed 70 TCRs of CD8+ TILs derived from two breast cancer patients and obtained 23 TCRs that reacted to tumor cells. Surprisingly, although two TCRs were HLA class I-restricted, the remaining 21 TCRs were non-HLA-restricted. Thus, c-FIT can be applied for monitoring multiple conventional and unconventional antigen-specific killer T cells in TILs, leading to the development of new designs for more effective T-cell-based immunotherapies.

A Survey of Near-Miss Dispensing Errors in Hospital Pharmacies in Japan: DEPP-J Study-Multi-Center Prospective Observational Study-

The aim of this study was to determine the proportion of near-miss dispensing errors in hospital pharmacies in Japan. A prospective multi-center observational study was conducted between December 2018 and March 2019. The primary objective was to determine the proportion of near-miss dispensing errors in hospital pharmacy departments. The secondary objective was to determine the predictive factors for near-miss dispensing errors using multiple logistic regression analysis. The study was approved by the ethical committee at The Institute of Medical Sciences, University of Tokyo, Japan. A multi-center prospective observational study was conducted in 20 hospitals comprising 8862 beds. Across the 20 hospitals, we assessed data from 553 pharmacists and 53039 prescriptions. A near-miss dispensing error proportion of 0.87% (n = 461) was observed in the study. We found predictive factors for dispensing errors in day-time shifts: a higher number of drugs in a prescription, higher number of quantified drugs, such as liquid or powder formula, in a prescription, and higher number of topical agents in a prescription; but we did not observe for career experience level for clinical pharmacists. For night-time and weekend shifts, we observed a negative correlation of near-miss dispensing errors with clinical pharmacist experience level. We found an overall incidence of near-miss dispensing errors of 0.87%. Predictive factors for errors in night-time and weekend shifts was inexperienced pharmacists. We recommended that pharmacy managers should consider education or improved work flow to avoid near-miss dispensing errors by younger pharmacists, especially those working night or weekend shifts.

Indications for SARS-CoV-2 nucleic acid amplification test for areas with low endemicity.

INTRODUCTION: The optimal indication for the nucleic acid amplification test (NAAT) in areas with low endemicity for coronavirus disease 2019 (COVID-19) is unclear. This study aimed to identify patients who should undergo the NAAT for COVID-19 diagnosis. METHODS: We retrospectively analyzed the clinical data of patients with suspected COVID-19 who underwent NAAT between October 5, 2020, and May 31, 2021 in our institution. RESULTS: A total of 1238 patients were enrolled and NAAT positive results were observed in 40 patients (3.2%). The NAAT positivity rate was 34.3% (23/67) in patients with a history of close contact and 1.5% (17/1171) in patients without a history of close contact. Olfactory/gustatory dysfunction and a history of stay in other prefectures were independent risk factors of COVID-19 in patients without a history of close contact. On the other hand, the NAAT positivity rate was only 0.7% (8/1073) in patients without olfactory/gustatory dysfunction and a history of stay in other prefectures. Among them, the group without respiratory symptoms/sign had only one NAAT-positive case (0.1%: 1/1073). CONCLUSIONS: This study revealed that a history of close contact, olfactory/gustatory dysfunction, and a history of stay in other prefectures are key eligibility criteria for NAAT in areas with relatively few patients with COVID-19. On the other hand, NAAT may not be necessary in cases without all of these factors and respiratory symptoms/sign.

Relationship between T cell receptor clonotype and PD-1 expression of tumor-infiltrating lymphocytes in colorectal cancer.

Adoptive T cell therapy using tumor-specific T cells or TCR-modified T cells is a promising next-generation immunotherapy. The major source of tumor-reactive T cells is PD-1+ tumor-infiltrating lymphocytes (TILs). In contrast, PD-1- TILs have received little attention. Here, we analyzed the TCR-ß repertoires of PD-1- and PD-1+ CD8+ TILs derived from colorectal cancer and breast cancer. Approximately 40-60% of the PD-1+ population consisted of oligoclonal populations in both colorectal cancer and breast cancer. In contrast, approximately 37% of the PD-1- population consisted of an oligoclonal population in colorectal cancer, whereas 14% of them were oligoclonal in breast cancer. In colorectal cancer, the TCR repertoires of PD-1- CD8+ TILs and PD-1+ CD8+ TILs hardly overlapped. Interestingly, clonally expanded CD8+ TILs in primary tumors and the metastases expressing the same clonotypic TCR showed the same phenotype regarding the PD-1-expression. These results suggest that the intrinsic properties of TCRs determine the fate of TILs in terms of whether they become PD-1+ or PD-1- in the tumor microenvironment. Further functional analysis of TCRs in TILs will allow us to better understand the regulatory mechanisms for PD-1 expression on TILs and may contribute to tumor immunotherapy.

Risk Prediction Model for Incident Atrial Fibrillation in a General Japanese Population　- The Hisayama Study.

BACKGROUND: The risk prediction of incident atrial fibrillation (AF) is useful to prevent AF and its complications. The aim of this study is to develop a new risk prediction model for incident AF using the prospective longitudinal data from a general Japanese population.Methods and Results:A total of 2,442 community-dwelling AF-free residents aged ≥40 years were followed up from 1988 to 2012 (46,422 person-years). The development of AF was confirmed by a standard 12-lead electrocardiogram at repeated health examinations and by medical records at clinics or hospitals. The risk prediction model for incident AF was developed using a Cox proportional hazards model. During the follow up, 230 AF events were confirmed. Age, sex, systolic blood pressure, waist circumference, estimated glomerular filtration rate, abnormal cardiac murmur, high R-wave amplitude, and arrhythmia other than AF were selected for inclusion in the model. This model showed good discrimination (Harrell's c statistics: 0.785) and calibration (Greenwood-Nam-D'Agostino test: P=0.87) for AF risk at 10 years. CONCLUSIONS: The new risk prediction model showed good performance on the individual risk assessment of the future onset of AF in a general Japanese population. As this model included commonly used clinical parameters, it may be useful for determining the requirements for the careful evaluation of AF, such as frequent electrocardiogram examinations in clinical settings, and subsequent reductions in the risk of AF-related complications.

Serum High-Sensitivity C-Reactive Protein Levels and the Development of Atrial Fibrillation in a General Japanese Population　- The Hisayama Study.

BACKGROUND: Atrial fibrillation (AF) is a common arrhythmia in the elderly, and causes complications such as cardioembolic stroke. Serum high-sensitivity C-reactive protein (hs-CRP), a marker of systemic inflammation, has been reported to be a risk factor for developing AF in Western countries. However, few community-based studies have examined this issue in general Asian populations.Methods and Results:A total of 2,510 community-dwelling Japanese participants aged ≥40 years without a history of AF were divided into 4 groups according to the sex-specific quartiles of serum hs-CRP concentrations (Q1, lowest and Q4, highest) and followed up for 24 years. The hazard ratios and their 95% confidence intervals for the development of AF were estimated using a Cox proportional hazards model. During the follow up, 234 subjects developed AF. The risk of AF increased significantly with elevating serum hs-CRP levels after adjustment for potential confounding factors (hazard ratio [95% confidence interval], Q1, 1.00 [reference]; Q2, 1.26 [0.83-1.92]; Q3, 1.77 [1.18-2.66]; and Q4, 1.89 [1.24-2.86]; P for trend <0.001). CONCLUSIONS: The study findings suggest that elevated serum hs-CRP levels are an independent risk factor for the development of AF in a general Japanese population.

Urinary N-terminal pro-B-type natriuretic peptide as a biomarker for cardiovascular events in a general Japanese population: the Hisayama Study.

BACKGROUND: Epidemiological evidence has shown that serum N-terminal pro-brain natriuretic peptide (NT-proBNP) concentrations, a diagnostic biomarker for heart failure, are positively associated with cardiovascular risk. Since NT-proBNP in serum is excreted in urine, it is hypothesized that urinary NT-proBNP concentrations are correlated with serum concentrations and linked with cardiovascular risk in the general population. METHODS: A total of 3060 community-dwelling residents aged ≥ 40 years without history of cardiovascular disease (CVD) were followed up for a median of 8.3 years (2007-2015). Serum and urinary concentrations of NT-proBNP at baseline were compared. The hazard ratios (HRs) and their 95% confidence intervals (CIs) for the association between NT-proBNP concentrations and the risk of developing CVD were computed using the Cox proportional hazards model. RESULTS: The median values (interquartile ranges) of serum and urinary NT-proBNP concentrations at baseline were 56 (32-104) pg/mL and 20 (18-25) pg/mL, respectively. There was a strong quadratic correlation between the serum and urinary concentrations of NT-proBNP (coefficient of determination [R2] = 0.72): urinary concentrations of 20, 27, and 43 pg/mL were equivalent to serum concentrations of 55, 125, and 300 pg/mL, respectively. During the follow-up period, 170 subjects developed CVD. The age- and sex-adjusted risk of CVD increased significantly with higher urinary NT-proBNP levels (P for trend < 0.001). This association remained significant after adjustment for traditional cardiovascular risk factors (P for trend = 0.009). The multivariable-adjusted risk of developing CVD almost doubled in subjects with urinary NT-proBNP of ≥ 43 pg/mL as compared to those with urinary NT-proBNP of ≤ 19 pg/mL (HR 2.07, 95% CI 1.20-3.56). CONCLUSIONS: The present study demonstrated that urinary NT-proBNP concentrations were well-correlated with serum concentrations and were positively associated with cardiovascular risk. Given that urine sampling is noninvasive and does not require specially trained personnel, urinary NT-proBNP concentrations have the potential to be an easy and useful biomarker for detecting people at higher cardiovascular risk.

[Two Cases of Hereditary Breast Cancer in Which Genetic Counseling Was Useful].

INTRODUCTION: The importance of genetic counseling has been noted for hereditary breast cancer. We report the cases of two 32-year-old woman, unmarried, BRCA mutation-positive patients. CASES: In Case 1, the chief complaint was awareness of a right breast mass. There was a family history of breast cancer(3 previous cases in the family). The BRCA2 mutation was positive during the BRACAnalysis test. Right breast mastectomy and axillary dissection were performed. Genetic counseling was performed postoperatively, and her eggs were cryopreserved. In Case 2, the chief complaint was calcification of the right breast. She also had a family history of breast cancer(3 previous cases in the family). The BRCA1 mutation was positive during the BRACAnalysis test. Nipple-sparing mastectomy and sentinel lymph node biopsy were performed, and the pathological diagnosis was DCIS. After genetic counseling, she did not desire the cryopreservation of her eggs. DISCUSSION: In these cases, the histological type, stage, subtype, BRCA mutation site, etc., were different from each other, and detailed support by genetic counseling was taken according to each medical condition such as surgery and postoperative adjuvant therapy.

Risk factors for postoperative pneumonia after general and digestive surgery: a retrospective single-center study.

PURPOSE: Pneumonia is the second-most common complication in postoperative patients and is associated with significant morbidity and high costs of care. We aimed to determine the risk factors for pneumonia after general and digestive surgery. METHODS: The medical records of 1,016 patients who underwent general and digestive surgery between January 2016 and March 2019 in our hospital were reviewed. RESULTS: Of the 1,016 patients, 67 (6.6%) developed postoperative pneumonia. The multivariate analysis showed that significant predictors of postoperative pneumonia were a poor Eastern Cooperative Oncology Group performance status (ECOG-PS), low forced vital capacity and low forced expiratory volume in one second in the spirometry test, malnutrition (low serum albumin levels and low controlling nutritional status scores and prognostic nutritional index [PNI] values), esophagectomy, upper gastrointestinal surgery, and nonlaparoscopic surgery. Of these factors, the combination of PNI and ECOG-PS clearly stratified patients into low-, intermediate-, and high-risk groups with respect to developing postoperative pneumonia (area under the curve: 0.709). CONCLUSIONS: Although postoperative pneumonia is associated with many clinical variables, active medical intervention for the prevention of pneumonia in patients with multiple risk factors can improve the postoperative course. In particular, perioperative nutritional care may prevent postoperative pneumonia in patients with malnutrition and a poor PS.

Clinical Relevance of a Candidate Stem Cell Marker, p75 Neurotrophin Receptor (p75NTR) Expression in Circulating Tumor Cells.

Despite advances in its diagnosis and multimodal therapies, the prognosis of esophageal squamous cell carcinoma (ESCC) patients remains poor, because of high incidences of metastasis . Recent reports suggested that circulating tumor stem cells (CTSCs), rather than circulating tumor cells (CTCs), were more accurate diagnostic marker for metastasis, because tumor stem cells or cancer stem cells (CSCs) are more responsible for metastasis through processes such as epithelial mesenchymal transition (EMT) and tumor initiation. A neurotrophin receptor p75 (p75NTR) is expressed in a candidate CSC s in ESCC, which possess enhanced tumorigenicity along with strong expression of EMT-related genes. Our recent report using two-color flow cytometry demonstrated that CTC counts based on a combined expression of epithelial cell adhesion molecule (EpCAM) and p75NTR was significantly higher in peripheral blood samples of ESCC patients than healthy controls. In addition, EpCAM + p75NTR+, but not EpCAM + p75NTR- CTC counts, correlated with clinically diagnosed distant metastasis and pathological venous invasion in surgically resected primary ESCC tumors. Malignant cytology of the isolated EpCAM + p75NTR+ cells was microscopically confirmed as well. These results demonstrated that EpCAM + p75NTR+ CTC count was a more accurate diagnostic marker than EpCAM+ CTC count, suggesting the highly metastatic potential of CTCs with p75NTR expression.Investigation using the isolated EpCAM + p75NTR+ CTCs to assess their stem cell properties may shed light on their roles in tumor metastasis in ESCC.Further investigations based on large-scale prospective studies with long term follow up may provide us with evidences for its clinical use.

The expression of microRNA 574-3p as a predictor of postoperative outcome in patients with esophageal squamous cell carcinoma.

BACKGROUND: Despite advances in radical esophagectomies and adjuvant therapy, the postoperative prognosis in esophageal squamous cell carcinoma (ESCC) patients remains poor. The aim of this study was to identify a molecular signature to predict postoperative favorable outcomes in patients with ESCC. METHODS: As a training data set, total RNA was extracted from formalin-fixed paraffin-embedded samples of surgically removed specimens from 19 ESCC patients who underwent curative esophagectomy. The expression of microRNA (miRNA) was detected using a miRNA oligo chip on which 885 genes were mounted. As a validation data set, we obtained frozen samples of surgically resected tumors from 12 independent ESCC patients and the expression of miR-574-3p was detected by quantitative real-time PCR. RESULTS: Our microarray analysis in the training set patients identified three miRNAs (miR-574-3p, miR-106b, and miR-1303) and five miRNAs (miR-1203, miR-1909, miR-204, miR-371-3p, miR-886-3p) which were differentially expressed between the patients with (n = 14) and without (n = 5) postoperative tumor relapse (p < 0.01 and p < 0.05, respectively). Higher expression of miR-574-3p, which showed the most significant association with non-relapse (p = 0.001), was associated with favorable overall survival (p = 0.016). Real-time PCR experiments on the validation set patients confirmed that higher expression of miR-574-3p was associated with non-tumor relapse (p = 0.029) and better overall survival (p = 0.004). CONCLUSIONS: Our results suggest that the aberrant expression of the miRNAs identified in this study plays key roles in the progression of ESCC. miR-574-3p was suggested to have a tumor suppressor effect, and thus, to be a predictor of postoperative outcome in patients with ESCC.

Detection of circulating tumor cells by p75NTR expression in patients with esophageal cancer.

BACKGROUND: The p75 neurotrophin receptor (p75NTR) is a cancer stem cell (CSC) marker in esophageal squamous cell carcinoma (ESCC). This study aimed to assess the use of p75NTR in detecting circulating tumor cells (CTCs) in ESCC. METHODS: Peripheral blood mononuclear cell expression of epithelial cell adhesion molecule (EpCAM) and p75NTR was detected in 23 ESCC patients (13 received chemo- or chemoradiotherapy and 10 received curative surgery) and 10 healthy controls by flow cytometry. RESULTS: EpCAM+p75NTR+ cell counts (average±SD) were significantly higher in patients (n=23, 16.0±18.3) compared to controls (n=10, 0.4±0.9, p=0.013). The sensitivity and specificity to differentiate ESCC patients from controls were 78.3 and 100% (cut-off value 4.0), respectively. EpCAM+p75NTR+, but not EpCAM+p75NTR- cell counts, correlated with clinically diagnosed distant metastasis (n=13, p=0.006) and pathological venous invasion in resected primary tumors (n=10, p=0.016). Malignant cytology was microscopically confirmed in isolated EpCAM+p75NTR+ cells with immunocytochemical double staining. CONCLUSIONS: p75NTR is suggested to be a useful marker for clinically significant CTCs, which exhibit highly metastatic features in ESCC.

Carbon ion radiotherapy for desmoid tumor of the abdominal wall: a case report.

BACKGROUND: Desmoid tumors, which are associated with familial adenomatous polyposis (FAP), tend to occur frequently in the abdominal wall and mesentery. Currently, there are no recognized treatments other than surgery, and frequent surgeries result in gastrointestinal obstructions and functional gastrointestinal disorders. CASE PRESENTATION: After surgery that was performed on a 39-year-old patient with FAP, we performed a second tumor excision which was the procedure used for frequently occurring mesenteric desmoid tumors. It was determined that the enlarged tumor would be difficult to operate on through an abdominal incision. Subsequently, the carbon ion radiotherapy of 50 Gy was then performed on the patient. Three years later, the tumor still remains reduced in size. In addition, we have not observed any negative effect on the digestive tract. CONCLUSIONS: This is the first instance that the carbon ion radiotherapy has been effective for the unresected desmoid tumor, and it is believed that this will become the one effective option for the treatment of desmoid tumors.

Functional outcome assessment of swallowing (FOAMS) scoring and videofluoroscopic evaluation of perioperative swallowing rehabilitation in radical esophagectomy.

PURPOSE: Oropharyngeal swallowing dysfunction following esophagectomy has been associated with the surgical disruption of muscle strength and flexibility of the oropharyngeal structures. We assessed the value of perioperative swallowing rehabilitation (SR) in patients who underwent radical esophagectomy. METHODS: We instituted routine perioperative SR for patients with esophageal cancer and retrospectively compared postoperative swallowing function between the patients who received (n = 12) vs. those who did not receive (n = 14) SR. RESULTS: The average duration of pre- and postoperative SR was 23.0 and 26.0 days, respectively. Preoperatively, the functional outcome assessment of the swallowing (FOAMS) score was 7 (full marks) in all 26 patients, whereas the average score at hospital discharge was 6.3 vs. 5.5 in the patients who received vs. those who did not receive SR, respectively (p = 0.049). Videofluoroscopic examination (n = 12) demonstrated that the maximum superior excursion of hyoid bone increased significantly with preoperative SR (p = 0.030), as well as postoperative SR (p = 0.046). However, perioperative SR did not reduce the incidence of postoperative aspiration pneumonia or the duration of hospital stay. CONCLUSIONS: Swallowing function after radical esophagectomy was improved by perioperative SR; however, further investigations are needed to assess the clinical significance of SR in reducing surgical complications.

Endoscopic thyroidectomy using the EZ-VANS method.

PURPOSE: Several video-assisted and robotic surgery techniques have been reported for resection of the thyroid and parathyroid glands. Our institute has started performing endoscopic thyroidectomy using the Lap-protector and E·Z-access system, referred to as E·Z-access using video-assisted neck surgery (EZ-VANS). In this report, we evaluate the safety and efficacy of this technique. METHODS: From January 2007 to September 2014, 110 patients underwent resection of a primary thyroid tumor, 73 who underwent a cervical collar incision (the Open group) and 37 underwent EZ-VANS (the EZ-VANS group). RESULTS: The average operating time was 159 and 172 min in the Open group and EZ-VANS group, respectively; the amount of blood loss was 46.5 and 54.7 ml, respectively; and the length of hospital stay after surgery was 4.3 and 5.2 days, respectively, with no significant differences observed between the two groups. The learning curve for the EZ-VANS technique was shorter than for open surgery. CONCLUSIONS: We confirmed that the EZ-VANS technique is a safe and useful method for resection of benign and early malignant thyroid tumors.

Crystal structure of a "loopless" GH19 chitinase in complex with chitin tetrasaccharide spanning the catalytic center.

DESCRIPTIONS: The structure of a GH19 chitinase from the moss Bryum coronatum (BcChi-A) in complex with the substrate was examined by X-ray crystallography and NMR spectroscopy in solution. The X-ray crystal structure of the inactive mutant of BcChi-A (BcChi-A-E61A) liganded with chitin tetramer (GlcNAc)4 revealed a clear electron density of the tetramer bound to subsites -2, -1, +1, and +2. Individual sugar residues were recognized by several amino acids at these subsites through a number of hydrogen bonds. This is the first crystal structure of GH19 chitinase liganded with oligosaccharide spanning the catalytic center. NMR titration experiments of chitin oligosaccharides into the BcChi-A-E61A solution showed that the binding mode observed in the crystal structure is similar to that in solution. The C-1 carbon of -1 GlcNAc, the Oε1 atom of the catalytic base (Glu70), and the Oγ atom of Ser102 form a "triangle" surrounding the catalytic water, and the arrangement structurally validated the proposed catalytic mechanism of GH19 chitinases. The glycosidic linkage between -1 and +1 sugars was found to be twisted and under strain. This situation may contribute to the reduction of activation energy for hydrolysis. The complex structure revealed a more refined mechanism of the chitinase catalysis.