Novel predictive approaches for drug-induced convulsions in non-human primates using machine learning and heart rate variability analysis.

Drug-induced convulsions are a major challenge to drug development because of the lack of reliable biomarkers. Using machine learning, our previous research indicated the potential use of an index derived from heart rate variability (HRV) analysis in non-human primates as a biomarker for convulsions induced by GABAA receptor antagonists. The present study aimed to explore the application of this methodology to other convulsants and evaluate its specificity by testing non-convulsants that affect the autonomic nervous system. Telemetry-implanted males were administered various convulsants (4-aminopyridine, bupropion, kainic acid, and ranolazine) at different doses. Electrocardiogram data gathered during the pre-dose period were employed as training data, and the convulsive potential was evaluated using HRV and multivariate statistical process control. Our findings show that the Q-statistic-derived convulsive index for 4-aminopyridine increased at doses lower than that of the convulsive dose. Increases were also observed for kainic acid and ranolazine at convulsive doses, whereas bupropion did not change the index up to the highest dose (1/3 of the convulsive dose). When the same analysis was applied to non-convulsants (atropine, atenolol, and clonidine), an increase in the index was noted. Thus, the index elevation appeared to correlate with or even predict alterations in autonomic nerve activity indices, implying that this method might be regarded as a sensitive index to fluctuations within the autonomic nervous system. Despite potential false positives, this methodology offers valuable insights into predicting drug-induced convulsions when the pharmacological profile is used to carefully choose a compound.

Comprehensive Cardiac Rehabilitation Following Acute Myocardial Infarction Improves Clinical Outcomes Regardless of Exercise Capacity.

BACKGROUND: Reduced exercise capacity is a prognostic indicator of adverse outcomes in patients with acute myocardial infarction (AMI). However, few studies have evaluated the effectiveness of comprehensive cardiac rehabilitation (CR) in this population. This study aimed to clarify the efficacy of comprehensive CR in patients with AMI and reduced exercise capacity.Methods and Results: This cohort study included 610 patients with AMI who underwent percutaneous coronary intervention. Major adverse cardiovascular events (MACE) were compared between patients who participated in comprehensive outpatient CR for 150 days (CR group; n=430) and those who did not (non-CR group; n=180). During the mean (±SD) follow-up period of 6.1±4.0 years, the CR group exhibited a lower incidence of MACE (log-rank P=0.002). Multivariable analysis revealed that Killip classification, diuretics at discharge, and participation in comprehensive CR were independently associated with MACE. The CR group was further divided into 2 groups, namely reduced exercise capacity (% predicted peak V̇O2<80%; n=241) and preserved exercise capacity (≥80%; n=147), based on the initial cardiopulmonary exercise test. Despite distinct exercise capacities, the incidence of MACE was comparable and physical parameters improved similarly after comprehensive CR in both groups. CONCLUSIONS: Comprehensive CR in patients with AMI effectively reduced the incidence of MACE regardless of initial exercise capacity. Cardiologists should actively encourage patients with low exercise capacity to participate in comprehensive CR.

Post-treatment Neutrophil/Lymphocyte Ratio Is a Prognostic Factor in Head and Neck Cancers Treated With Nivolumab.

Background/Aim: Inflammation and nutrition-based biomarkers, such as the neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), lymphocyte/monocyte ratio (LMR), C-reactive protein/albumin ratio (CAR), prognostic nutritional index (PNI), systemic immune inflammation index (SII), and systemic inflammation response index (SIRI), have prognostic value for several types of malignancies. Markers that precisely reflect the prognosis of patients with head and neck cancers (HNCs) treated with immune-checkpoint inhibitors remain unclear. This retrospective study aimed to investigate the prognostic value of hematological markers before and after treatment with nivolumab in patients with recurrent or metastatic HNC (RM-HNC). Patients and Methods: We evaluated the clinical data of 44 patients with recurrent/metastatic head and neck squamous cell carcinoma treated with nivolumab between April 2017 and April 2023 at Shinshu University Hospital. Values of hematological biomarkers (NLR, LMR, PLR, CAR, PNI, SII, and SIRI) were calculated before and 4-6 weeks after nivolumab initiation. Receiver operating characteristic curves were constructed to determine the cutoff values of pre- and post-treatment markers for overall survival (OS) and progression-free survival (PFS). Results: Among all pre- and post-treatment markers, post-treatment NLR showed the highest area under the curve (AUC=0.702). A high post-treatment NLR (cutoff value, 4.01) was associated with a poor OS (p=0.027) and a tendency for shorter PFS (p=0.117). Multivariate analysis showed that a high post-treatment NLR was significantly associated with poor OS (p=0.026). Conclusion: A high post-treatment NLR was associated with poor response to nivolumab in head and neck cancers.

A Spatial Transcriptome Reveals Changes in Tumor and Tumor Microenvironment in Oral Cancer with Acquired Resistance to Immunotherapy.

Although anti-programmed death-1 (PD-1) antibody therapy improves the prognosis in patients with head and neck squamous cell carcinoma (HNSCC), some patients exhibit disease progression even after showing a good response to the treatment initially because of acquired resistance. Here, we aimed to reveal the dynamic changes in the tumor and tumor microenvironment (TME) in a 77-year-old man diagnosed with oral squamous cell carcinoma who developed acquired resistance after the administration of nivolumab using spatial transcriptomics. The results showed that, before immunotherapy, the activated pathways in the tumor area were mainly related to the cancer immune system, including antigen processing cross-presentation, interferon-gamma signaling, and the innate immune system. After immunotherapy, the activated pathways were mainly related to epigenetic modification, including RMTs methylate histone arginine and HDAC deacetylates histones. Before immunotherapy, the activated pathways in the TME were mainly related to the metabolism of proteins, including SRP-dependent co-translational protein targeting the membrane. After immunotherapy, the activated pathways in the TME were related to sensory perception and signal transduction. Our study revealed that epigenetic-modification-related pathways were mainly activated after establishing acquired resistance, suggesting that epigenetic modification in the tumor may prevent cancer immune system activation via the anti-PD-1 antibody.

Remote Cardiac Rehabilitation With Wearable Devices.

Although cardiac rehabilitation (CR) has been shown to improve exercise tolerance and prognosis in patients with cardiovascular diseases, there remains low participation in outpatient CR. This may be attributed to the patients' busy schedules and difficulty in visiting the hospital due to distance, cost, avoidance of exercise, and severity of coronary disease. To overcome these challenges, many countries are exploring the possibility of remote CR. Specifically, there is increasing attention on the development of remote CR devices, which allow transmission of vital information to the hospital via a remote CR application linked to a wearable device for telemonitoring by dedicated hospital staff. In addition, remote CR programs can support return to work after hospitalization. Previous studies have demonstrated the effects of remote CR on exercise tolerance. However, the preventive effects of remote CR on cardiac events and mortality remain controversial. Thus, safe and effective remote CR requires exercise risk stratification for each patient, telenursing by skilled staff, and multidisciplinary interventions. Therefore, quality assurance of telenursing and multi-disciplinary interventions will be essential for remote CR. Remote CR may become an important part of cardiac management in the future. However, issues such as cost-effectiveness and insurance coverage still persist.

Utility of Cardiac Rehabilitation for Long-Term Outcomes in Patients with Hospital-Acquired Functional Decline after Cardiac Surgery: A Retrospective Study.

Hospital-acquired functional decline is an important outcome that affects the long-term prognosis of patients after cardiac surgery. Phase II cardiac rehabilitation (CR) for outpatients is expected to improve prognosis; however, this is not clear in patients with hospital-acquired functional decline after cardiac surgery. Therefore, this study evaluated whether phase II CR improved the long-term prognosis of patients with hospital-acquired functional decline after cardiac surgery. This single-center, retrospective observational study included 2371 patients who required cardiac surgery. Hospital-acquired functional decline occurred in 377 patients (15.9%) after cardiac surgery. The mean follow-up period was 1219 ± 682 days in all patients, and there were 221 (9.3%) cases with major adverse cardiovascular events (MACE) after discharge during the follow-up period. The Kaplan-Meier survival curves indicated that hospital-acquired functional decline and non-phase II CR was associated with a higher incidence of MACE than other groups (log-rank, p < 0.001), additionally exhibiting prognosticating MACE in multivariate Cox regression analysis (HR, 1.59; 95% CI, 1.01-2.50; p = 0.047). Hospital-acquired functional decline after cardiac surgery and non-phase II CR were risk factors for MACE. The participation in phase II CR in patients with hospital-acquired functional decline after cardiac surgery could reduce the risk of MACE.

Exploring the Frailty Components Related to Hospitalization-Associated Disability in Older Patients After Cardiac Surgery Using a Comprehensive Frailty Assessment.

BACKGROUND: We investigated the components of frailty associated with hospitalization-associated disability (HAD) after cardiac surgery.Methods and Results: This retrospective, observational study evaluated 1,446 older patients after elective cardiac surgery at the Sakakibara Heart Institute. We examined the association between HAD and 7 domains of frailty assessed by the Kihon Checklist. HAD was defined as a decline in the ability to perform activities of daily living (ADL) between admission and discharge, as assessed by the Barthel Index. Logistic regression and decision tree analysis were used to identify associations between the number and type of frailty components and HAD. Of the 1,446 patients, 190 were excluded, and 90 (7%) developed HAD. An increase in the number of frailty components was a risk factor for HAD (odds ratio: 1.88, 95% confidence interval: 1.62-2.17). Decision tree analysis identified physical functional decline, depression, and cognitive dysfunction as factors associated with HAD. The incidence of HAD was highest in cases of physical functional decline (21%) and lowest for cases in which the 3 aforementioned factors were absent (2.8%). CONCLUSIONS: An increased number of frailty factors increased the risk of HAD and the findings also reaffirmed the importance of a comprehensive assessment to evaluate the risk of HAD, including evaluation of physical function, cognitive function, and depression.

Prognostic significance of pre- and post-treatment hematological biomarkers in patients with head and neck cancer treated with chemoradiotherapy.

This study aimed to investigate the prognostic value of hematological biomarkers measured before and after treatment in patients with head and neck cancer (HNC). This study reviewed 124 patients with HNC who received chemoradiotherapy. Hematological biomarkers assessed before and after treatment were investigated. The pretreatment C-reactive protein/albumin ratio (pre-CAR) and post-treatment prognostic nutritional index (post-PNI) showed the highest area under the curve with cutoff values of 0.0945 and 34.9, respectively. Patients in the high pre-CAR group showed significantly worse prognosis than those in the low pre-CAR group with respect to the progression-free survival (PFS) (3-year PFS: 44.8% vs. 76.8%, p < 0.001) and overall survival (OS) (3-year OS: 65.8% vs. 94.0%, p < 0.001). Patients in the low post-PNI group showed significantly worse prognosis than those in the high post-PNI group with respect to the PFS (3-year PFS: 58.6% vs. 77.4%, p = 0.013) and OS (3-year OS: 75.2% vs. 96.9%, p = 0.019). Multivariate analysis revealed that advanced N stage (p = 0.008), high pre-CAR (p = 0.024), and low post-PNI (p = 0.034) were significantly associated with poorer OS. We suggest that the evaluation of hematological markers before and after treatment is useful for predicting disease progression and survival.

Factors Related to Hospitalisation-Associated Disability in Patients after Surgery for Acute Type A Aortic Dissection: A Retrospective Study.

The in-hospital mortality rate among patients after surgery for acute type A aortic dissection (ATAAD) has improved chronologically. However, the relationship between the incidence of hospitalisation-associated disability (HAD) and acute cardiac rehabilitation in patients after surgery for ATAAD has not been reported. Therefore, this study evaluated factors related to HAD in patients after surgery for ATAAD. This single-centre retrospective observational study included 483 patients who required emergency surgery for ATAAD. HAD occurred in 104 (21.5%) patients following cardiovascular surgery. Factors associated with HAD were age (odds ratio [OR], 1.05; 95% confidence interval [CI], 1.02-1.09; p = 0.001), noninvasive positive pressure ventilation (NPPV; OR, 2.15; 95% CI, 1.10-4.19; p = 0.025), postoperative delirium (OR, 2.93; 95% CI, 1.60-5.37; p = 0.001), and timing of walking onset (OR, 1.29; 95% CI, 1.07-1.56; p = 0.008). Furthermore, a late walking onset was associated with a higher risk of developing HAD and more severe functional decline. Early rehabilitation based on appropriate criteria has possibility of preventing HAD.

Perioperative management of a patient with a giant thyroglossal duct cyst: a case report.

Thyroglossal duct cysts (TGDC) are the most common type of congenital neck masses, which generally present in young adults. We present a rare case of a giant TGDC in a 77-year-old patient who required atypical perioperative management. The patient presented with a large soft mass on his anterior neck. Computed tomography showed a lobulated cystic mass measuring 18 × 16 cm, extending from the tongue base to the inferior level of the clavicle. Because difficult intubation was expected, the cyst was punctured and most of the fluid was drained prior to surgery. The swelling of the tongue base was remarkably reduced, and intubation was performed safely. The cyst was extracted using the Sistrunk procedure and tracheotomy was performed. Histopathological examination confirmed the diagnosis of TGDC. Preoperative volume reduction of the cyst and tracheotomy should be considered for oral intubation and postoperative airway management, respectively, in patients with large TGDC.

Associations of body mass index and hospital-acquired disability with post-discharge mortality in older patients with acute heart failure.

OBJECTIVES: To investigate the effect of hospital-acquired disability (HAD) on all-cause mortality after discharge according to the body mass index (BMI) in older patients with acute decompensated heart failure. METHODS: We included 408 patients aged ≥ 65 years who were hospitalized for acute decompensated heart failure and had undergone an acute phase of cardiac rehabilitation at the Sakakibara Heart Institute between April 2013 and September 2015 (median age: 82 years, interquartile range (IQR): 76-86; 52% male). Patients were divided into three groups based on BMI at hospital admission: underweight (< 18.5 kg/m2), normal weight (18.5 to 25 kg/m2), and overweight (≥ 25 kg/m2). HAD was defined as a decrease of at least five points at discharge compared to before hospitalization according to the Barthel Index. RESULTS: The median follow-up period was 475 (IQR: 292-730) days, and all-cause mortality during the follow-up period was 84 deaths (21%). According to multivariate Cox regression analysis, being underweight (HR: 1.941, 95% CI: 1.134-3.321,P = 0.016) or overweight (HR: 0.371, 95% CI: 0.171-0.803,P = 0.012), with normal BMI as the reference, and HAD (HR: 1.857, 95% CI: 1.062-3.250,P = 0.030) were independently associated with all-cause mortality. Patients with HAD exhibited a significantly lower cumulative survival rate in the underweight group (P = 0.001) and tended to have a lower cumulative survival rate in the normal weight group (P = 0.072). HAD was not significantly associated with cumulative survival in the overweight group (P = 0.392). CONCLUSIONS: BMI and HAD independently predicted all-cause mortality after discharge in older patients with acute decompensated heart failure. Furthermore, HAD was significantly associated with higher all-cause mortality after discharge, especially in the underweight group.

Physical function examination at intensive care unit as predictive indicators for hospitalization-associated disability in patients after cardiovascular surgery.

BACKGROUND: Following cardiovascular surgery, patients are at high risk of requiring systemic management in the intensive care unit (ICU), resulting in hospitalization-associated disability (HAD). Predicting the risk of HAD during the postoperative course is important to prevent susceptibility to cardiovascular events. Assessment of physical function during the ICU stay may be useful as a prediction index but has not been established. METHODS: This prospective observational study conducted at a high-volume cardiovascular center included 236 patients (34% female; median age, 73 years) who required an ICU stay of at least 72 hours after surgery and underwent postoperative rehabilitation. HAD was defined as a decrease in the discharge Barthel index (BI) score of at least 5 points relative to the preadmission BI score. Physical Function ICU Test-scored (PFIT-s), Functional Status Score for the ICU (FSS-ICU), and Medical Research Council (MRC)-sumscore were used to assess physical function at ICU discharge. RESULTS: HAD occurred in 58 (24.6%) of the 236 patients following cardiovascular surgery. The cut-off points for HAD were 7.5 points for the PFIT-s (sensitivity 0.80, specificity 0.59), 24.5 points for the FSS-ICU (sensitivity 0.57, specificity 0.66), and 59.5 points for the MRC-sumscore (sensitivity 0.93, specificity 0.66). Multivariate logistic regression analysis revealed a PFIT-s of >7.5 points (odds ratio [OR], 4.84; 95% CI, 2.39-9.80; p < 0.001) and an MRC-sumscore of >59.5 points (OR, 2.43; 95% CI, 1.22-4.87; p = 0.012) as independent associated factors. CONCLUSIONS: We demonstrate that the PFIT-s and MRC-sumscore at ICU discharge may be helpful as a predictive indicator for HAD in patients having undergone major cardiovascular surgery.

Psoas Muscle Volume and Attenuation are Better Predictors than Muscle Area for Hospital Readmission in Older Patients after Transcatheter Aortic Valve Implantation.

OBJECTIVE: This study aimed to determine whether the psoas muscle volume (PMV) and its muscle attenuation (MA) are associated with hospital readmission after transcatheter aortic valve implantation (TAVI). METHOD: We included 113 older patients with aortic stenosis who underwent TAVI at Sakakibara Heart Institute (mean age 85 ± 5 years, 69% women). We measured PMV and psoas muscle area (PMA) as well as total muscle area (TMA) at the third lumbar vertebra using preoperative computed tomography (CT) images. The crude values of the PMV, PMA, and TMA were normalized by dividing by height squared. RESULTS: The median follow-up period was 724 days (interquartile range: 528-730 days), and there were 25 all-cause readmissions during the follow-up period (22% of all patients). In the multivariate Cox regression analysis adjusted for age, sex, and EuroSCORE II, the PMV and its MA and crude PMA were significantly associated with all-cause readmission [HR: 0.957 (0.930-0.985), p = 0.003, HR: 0.927 (0.862-0.997), p = 0.040], whereas the PMA and TMA and each MA were not (all p > 0.05). The groups with low PMV and MA had significantly higher incidences of all-cause readmission than that with high PMV and MA (log-rank test: p = 0.011). CONCLUSION: PMV and its MA measured from preoperative CT images were independent predictors of all-cause readmission in TAVI patients.

Hospitalization-Associated Disability After Cardiac Surgery in Elderly Patients　- Exploring the Risk Factors Using Machine Learning Algorithms.

Background: Hospitalization-associated disability (HAD) is associated with prolonged functional decline and increased mortality after discharge. Therefore, we examined the incidence and risk factors associated with HAD in elderly patients undergoing cardiac surgery in Japan. Methods and Results: We retrospectively examined 2,262 elderly patients who underwent elective cardiac surgery at Sakakibara Heart Institute. HAD was defined as a functional decline between time of admission and discharge measured by the Barthel Index. We analyzed clinical characteristics using machine learning algorithms to identify the risk factors associated with HAD. After excluding 203 patients, 2,059 patients remained, of whom 108 (5.2%) developed HAD after cardiac surgery. The risk factors identified were age, serum albumin concentration, estimated glomerular filtration rate, Revised Hasegawa's Dementia Scale, N-terminal pro B-type natriuretic peptide, vital capacity, preoperative Short Physical Performance Battery (SPPB) score, operation times, cardiopulmonary bypass times, ventilator times, length of postoperative intensive care unit stay, and postoperative ambulation start day. The highest incidence of HAD was found in patients with an SPPB score ≤9 and in those who started ambulation >6 days after surgery (76.9%). Conclusions: Several risk factors for HAD are components of frailty, suggesting that preoperative rehabilitation to reduce the risk of HAD is feasible. Furthermore, the association between HAD and a delayed start of ambulation reaffirms the importance of early mobilization and rehabilitation.

Prognostic impact of hospital-acquired disability in elderly patients with heart failure.

AIMS: Functional decline is associated with worse outcomes in patients with elderly heart failure (HF), but little is known about the prognostic impact of hospital-acquired disability (HAD) during hospital stay after acute HF. The present study examines the prognostic significance of HAD in the prediction of all-cause mortality in elderly patients who admitted for acute HF. METHODS AND RESULTS: This retrospective study was performed in 1941 elderly patients aged ≥65 years or older from the cardiovascular physiotherapy for acute HF patients in the Tokyo metropolitan area registry and excluded those who died in hospital. HAD was defined as any decline in the Barthel index (BI) before discharge compared with the BI within 1 month before hospital admission. The primary outcome of this study was all-cause death and HF readmission. A total of 565 (29%) deaths and 789 (41%) HF readmission occurred over a median follow-up period of 1.7 years. A total of 476 patients (25%) had HAD during hospital stay after acute HF. In multivariable analysis, HAD predicted all-cause death [hazard ratio (HR): 1.772; 95% confidence interval (CI): 1.450-2.167; P < 60; 0.001] and with risk of HF readmission (HR: 1.193; 95% CI: 1.005-1.416; P = 0.043) after adjusting for the Meta-analysis Global Group in Chronic Heart Failure risk score. CONCLUSIONS: Hospital-acquired disability is associated with an increased risk of all-cause death and readmission for HF in elderly patients with acute HF.

Comparative analysis using the draft genome sequence of California poppy (Eschscholzia californica) for exploring the candidate genes involved in benzylisoquinoline alkaloid biosynthesis.

Genome characterization of California poppy (Eschscholzia californica cv. "Hitoezaki"), which produces pharmaceutically important benzylisoquinoline alkaloids (BIAs), was carried out using the draft genome sequence. The numbers of tRNA and rRNA genes were close to those of the other plant species tested, whereas the frequency of repetitive sequences was distinct from those species. Comparison of the predicted genes with those of Amborella trichopoda, Nelumbo nucifera, Solanum lycopersicum, and Arabidopsis thaliana, and analyses of gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway indicated that the enzyme genes involved in BIA biosynthesis were highly enriched in the California poppy genome. Further comparative analysis using the genome information of Papaver somniferum and Aquilegia coerulea, both BIA-producing plants, revealed that many genes encoding BIA biosynthetic enzymes, transcription factors, transporters, and candidate proteins, possibly related to BIA biosynthesis, were specifically distributed in these plant species.

Over- and Under-sampling Approach for Extremely Imbalanced and Small Minority Data Problem in Health Record Analysis.

A considerable amount of health record (HR) data has been stored due to recent advances in the digitalization of medical systems. However, it is not always easy to analyze HR data, particularly when the number of persons with a target disease is too small in comparison with the population. This situation is called the imbalanced data problem. Over-sampling and under-sampling are two approaches for redressing an imbalance between minority and majority examples, which can be combined into ensemble algorithms. However, these approaches do not function when the absolute number of minority examples is small, which is called the extremely imbalanced and small minority (EISM) data problem. The present work proposes a new algorithm called boosting combined with heuristic under-sampling and distribution-based sampling (HUSDOS-Boost) to solve the EISM data problem. To make an artificially balanced dataset from the original imbalanced datasets, HUSDOS-Boost uses both under-sampling and over-sampling to eliminate redundant majority examples based on prior boosting results and to generate artificial minority examples by following the minority class distribution. The performance and characteristics of HUSDOS-Boost were evaluated through application to eight imbalanced datasets. In addition, the algorithm was applied to original clinical HR data to detect patients with stomach cancer. These results showed that HUSDOS-Boost outperformed current imbalanced data handling methods, particularly when the data are EISM. Thus, the proposed HUSDOS-Boost is a useful methodology of HR data analysis.

Mining of the Uncharacterized Cytochrome P450 Genes Involved in Alkaloid Biosynthesis in California Poppy Using a Draft Genome Sequence.

Land plants produce specialized low molecular weight metabolites to adapt to various environmental stressors, such as UV radiation, pathogen infection, wounding and animal feeding damage. Due to the large variety of stresses, plants produce various chemicals, particularly plant species-specific alkaloids, through specialized biosynthetic pathways. In this study, using a draft genome sequence and querying known biosynthetic cytochrome P450 (P450) enzyme-encoding genes, we characterized the P450 genes involved in benzylisoquinoline alkaloid (BIA) biosynthesis in California poppy (Eschscholzia californica), as P450s are key enzymes involved in the diversification of specialized metabolism. Our in silico studies showed that all identified enzyme-encoding genes involved in BIA biosynthesis were found in the draft genome sequence of approximately 489 Mb, which covered approximately 97% of the whole genome (502 Mb). Further analyses showed that some P450 families involved in BIA biosynthesis, i.e. the CYP80, CYP82 and CYP719 families, were more enriched in the genome of E. californica than in the genome of Arabidopsis thaliana, a plant that does not produce BIAs. CYP82 family genes were highly abundant, so we measured the expression of CYP82 genes with respect to alkaloid accumulation in different plant tissues and two cell lines whose BIA production differs to estimate the functions of the genes. Further characterization revealed two highly homologous P450s (CYP82P2 and CYP82P3) that exhibited 10-hydroxylase activities with different substrate specificities. Here, we discuss the evolution of the P450 genes and the potential for further genome mining of the genes encoding the enzymes involved in BIA biosynthesis.

Unraveling Additional O-Methylation Steps in Benzylisoquinoline Alkaloid Biosynthesis in California Poppy (Eschscholzia californica).

California poppy (Eschscholzia californica), a member of the Papaveraceae family, produces many biologically active benzylisoquinoline alkaloids (BIAs), such as sanguinarine, macarpine and chelerythrine. Sanguinarine biosynthesis has been elucidated at the molecular level, and its biosynthetic genes have been isolated and used in synthetic biology approaches to produce BIAs in vitro. However, several genes involved in the biosynthesis of macarpine and chelerythrine have not yet been characterized. In this study, we report the isolation and characterization of a novel O-methyltransferase (OMT) involved in the biosynthesis of partially characterized BIAs, especially chelerythrine. A search of the RNA sequence database from NCBI and PhytoMetaSyn for the conserved OMT domain identified 68 new OMT-like sequences, of which the longest 22 sequences were selected based on sequence similarity. Based on their expression in cell lines with different macarpine/chelerythrine profiles, we selected three OMTs (G2, G3 and G11) for further characterization. G3 expression in Escherichia coli indicated O-methylation activity of the simple benzylisoquinolines, including reticuline and norreticuline, and the protoberberine scoulerine with dual regio-reactivities. G3 produced 7-O-methylated, 3'-O-methylated and dual O-methylated products from reticuline and norreticuline, and 9-O-methylated tetrahydrocolumbamine, 2-O-methylscoulerine and tetrahydropalmatine from scoulerine. Further enzymatic analyses suggested that G3 is a scoulerine-9-O-methyltransferase for the biosynthesis of chelerythrine in California poppy. In the present study, we discuss the physiological role of G3 in BIA biosynthesis.

Efficient microbial production of stylopine using a Pichia pastoris expression system.

Stylopine is a protoberberine-type alkaloid that has potential biological activities. Based on the successful microbial production of (S)-reticuline, we attempted to produce stylopine from (S)-reticuline by the reaction of berberine bridge enzyme, cheilanthifoline synthase (CYP719A5), and stylopine synthase (CYP719A2). Biosynthetic enzyme expression was examined in a methanol-utilizing yeast (Pichia pastoris), and both a "consolidated" system with all genes expressed in one cell and a "co-culture" system with three cell lines that each express a single gene were examined. Although both systems efficiently converted reticuline to stylopine, the consolidated system was more rapid and efficient than the co-culture system. However, substrate-feeding experiments revealed a decrease in the conversion efficiency in the consolidated system during successive cultures, whereas the conversion efficiency in the co-culture system remained constant. Thus, the final amount of stylopine produced from reticuline after successive feedings in the co-culture system was more than 150 nmoles from 750 nmoles of (R, S)-reticuline (375 nmoles of (S)-reticuline). The advantages and drawbacks of the "consolidated" system and the "co-culture" system are discussed.