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The bone is the most common location of breast cancer metastasis. Bone metastasis causes pain and skeletal-related events (SREs), and affects the quality of life and survival of breast cancer patients. Bone-modifying agents, including bisphosphonates and denosumab, reduce the risk of SREs in these patients. Among bisphosphonates, zoledronic acid is preferred due to its higher efficacy. Denosumab is marginally more effective than zoledronic acid in reducing the frequency of SREs. Patients with renal impairment should receive zoledronic acid with caution due to nephrotoxicity, and denosumab is an alternative option in these patients. Osteonecrosis of the jaw is a rare but severe complication of both zoledronic acid and denosumab, and all patients should undergo dental examinations before and during treatment. Although these drugs can reduce the risk of SREs in bone metastasis, no significant improvement in survival has been noted. This study reviews the effects and adverse events of bone-modifying agents for the treatment of bone metastasis from breast cancer.

Abstract This study evaluated an oscillometric device (OD), Microlife WatchBP Office AFIB, and a hybrid manual auscultatory device (AD), Greenlight 300TM, to determine a suitable blood pressure (BP) measurement device for the Korea National Health and Nutrition Examination Survey in a mercury‐free context. Adhering to the 2018 Universal Standard's suggested consensus, the study involved 800 subjects (mean age 51.2 ± 17.5 years; 44.3% male), who underwent triplicate BP measurements following 5 min of rest in a randomized order (OD‐first: 398 participants; AD‐first: 402 participants). BP difference was calculated as OD value minus AD value, with results stratified by measurement sequence. The overall BP difference and tolerable error probability were ‐1.1 ± 6.5/‐2.6 ± 4.9 mmHg and 89.2%/92.5% for systolic/diastolic BP (SBP/DBP), respectively. Lin's concordance correlation coefficient was 0.907/0.844 for SBP/DBP (OD‐first/AD‐first: 0.925/0.892 for SBP, 0.842/0.845 for DBP). The overall agreement for hypertension (BP ≥ 140 and/or 90 mmHg) was 0.71 (p

Abstract Background Intratumoral heterogeneity (ITH) and tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) play important roles in tumor evolution and patient outcomes. However, the precise characterization of diverse cell populations and their crosstalk associated with PDAC progression and metastasis is still challenging. Methods We performed single-cell RNA sequencing (scRNA-seq) of treatment-naïve primary PDAC samples with and without paired liver metastasis samples to understand the interplay between ITH and TME in the PDAC evolution and its clinical associations. Results scRNA-seq analysis revealed that even a small proportion (22%) of basal-like malignant ductal cells could lead to poor chemotherapy response and patient survival and that epithelial-mesenchymal transition programs were largely subtype-specific. The clonal homogeneity significantly increased with more prevalent and pronounced copy number gains of oncogenes, such as KRAS and ETV1, and losses of tumor suppressor genes, such as SMAD2 and MAP2K4, along PDAC progression and metastasis. Moreover, diverse immune cell populations, including naï ve SELL hi regulatory T cells (Tregs) and activated TIGIT hi Tregs, contributed to shaping immunosuppressive TMEs of PDAC through cellular interactions with malignant ductal cells in PDAC evolution. Importantly, the proportion of basal-like ductal cells negatively correlated with that of immunoreactive cell populations, such as cytotoxic T cells, but positively correlated with that of immunosuppressive cell populations, such as Tregs. Conclusion We uncover that the proportion of basal-like subtype is a key determinant for chemotherapy response and patient outcome, and that PDAC clonally evolves with subtype-specific dosage changes of cancer-associated genes by forming immunosuppressive microenvironments in its progression and metastasis.

Abstract Many individuals have different blood pressure (BP) values in the office setting compared to that outside the office setting. Therefore, confirming hypertension based on office BP (OBP) measurement alone can lead to misdiagnosis and mistreatment. The limitations of OBP measurement have led to the complementary use of out-of-office BP measurements, including 24-hour ambulatory blood pressure monitoring (ABPM) and home blood pressure monitoring (HBPM). This review aims to describe when and how ABPM or HBPM can be used to accurately diagnose and treat hypertension. Both methods should be performed using validated automated oscillometric devices. To minimize user errors, ABPM should be performed using standard techniques, whereas HBPM requires patient education regarding proper BP measurements. ABPM provides short-term comprehensive information on BP, including daytime, nighttime, morning, and 24-h BP. Therefore, ABPM is recommended for the initial diagnosis of hypertension, assessment of BP phenotypes and circadian patterns, and detection of nocturnal hypertension, Furthermore, ABPM plays a critical role in confirming true resistant hypertension thereby excluding pseudo-resistant hypertension. However, it is not suitable for long-term follow-up of patients with hypertension. In contrast, HBPM involves multiple BP readings taken at specific times during the day and evening over a long period. Therefore, HBPM is recommended for diagnosing hypertension and assessing BP phenotypes. However, this method has limitations in measuring nocturnal BP and circadian BP patterns. HBPM is preferred over ABPM for the long-term follow-up of patients with hypertension. This approach improves patient adherence to treatment and ultimately enhances the rate of control of hypertension. Additionally, both methods play an important role in diagnosing and treating white coat hypertension during pregnancy. Consequently, out-of-office BP measurement is essential to prevent the misdiagnosis and mistreatment of hypertension. However, these two methods offer different information regarding the BP status of an individual, and they indeed show discrepancies in the diagnosis of hypertensive phenotypes. Therefore, it is crucial to understand the advantages and limitations of both ABPM and HBPM to ensure their appropriate use in clinical practice. Graphical Abstract

Abstract Although reports vary, the prevalence of true resistant hypertension and apparent treatment-resistant hypertension (aTRH) has been reported to be 10.3% and 14.7%, respectively. As there is a rapid increase in the prevalence of obesity, chronic kidney disease, and diabetes mellitus, factors that are associated with resistant hypertension, the prevalence of resistant hypertension is expected to rise as well. Frequently, patients with aTRH have pseudoresistant hypertension [aTRH due to white-coat uncontrolled hypertension (WUCH), drug underdosing, poor adherence, and inaccurate office blood pressure (BP) measurements]. As the prevalence of WUCH is high among patients with aTRH, the use of out-of-office BP measurements, both ambulatory blood pressure monitoring (ABPM) and home blood pressure monitoring (HBPM), is essential to exclude WUCH. Non-adherence is especially problematic, and methods to assess adherence remain limited and often not clinically feasible. Therefore, the use of HBPM and higher utilization of single-pill fixed-dose combination treatments should be emphasized to improve drug adherence. In addition, primary aldosteronism and symptomatic obstructive sleep apnea are quite common in patients with hypertension and more so in patients with resistant hypertension. Screening for these diseases is essential, as the treatment of these secondary causes may help control BP in patients who are otherwise difficult to treat. Finally, a proper drug regimen combined with lifestyle modifications is essential to control BP in these patients. Graphical Abstract

The purpose of the study was to identify subgroups of advanced cancer patients who experienced grade 3–4 toxicities as reported by their oncologists as well as identify the demographic, clinical, and treatment symptom characteristics as well as QoL outcomes associated with distinct profiles of each patient. A prospective, multicenter, observational study was conducted with advanced cancer patients of 15 different hospitals across Spain. After three months of systemic cancer treatment, participants completed questionnaires that evaluated psychological distress (BSI-18), quality of life (EORTC QLQ-C30) and fatigue (FAS). The most common tumor sites for the 557 cancer patients with a mean age of 65 years were bronchopulmonary, digestive, and pancreas. Overall, 19% of patients experienced high-grade toxicities (grade 3–4) during treatment. Patients with recurrent advanced cancer, with non-adenocarcinoma cancer, undergoing chemotherapy, and a showing deteriorated baseline status (ECOG > 1) were more likely to experience higher toxicity. Patients who experienced grade 3–4 toxicities during cancer treatment had their treatment suspended in 59% of the cases. Additionally, 87% of the patients had a dose adjustment or a cycle delayed in their treatment due to a high risk of dying during treatment. Future research should focus on identifying interventions to reduce high-grade toxicities and improve quality of life in cancer patients.

Abstract Accurate blood pressure (BP) measurement is crucial for hypertension detection and management. The Korea National Health and Nutrition Examination Survey (KNHANES) assesses the health of Koreans using representative cross-sectional data. BP measurements were historically done with mercury sphygmomanometers for participants aged ≥10 years. However, KNHANES transitioned to Greenlight 300TM (mercury-free auscultatory device) in 2020 for participants aged ≥6 years and used dual devices (Microlife WatchBP Office AFIB and Greenlight) in 2021-2022. To ensure consistency, KNHANES will adopt Microlife as the unified BP device with Greenlight for device validation from 2023. Under the new protocol, participants aged ≥6 years will have their BP measured three times at 30-second intervals after a 5-minute rest under ambient temperature (20-25℃) and noise ≤65 dB. The average of the 2nd and 3rd readings will be used as the representative BP value. The quality control (QC) program involves four trained examiners passing the “quality control and assurance of BP measurement program” three times annually, and undergoing “video monitoring of weekly calibration process” once a year. Additionally, the QC team will conduct “on-site evaluations of BP measurement” at mobile examination centers three times a year. A Five-Step QC process for BP devices was also developed. This document outlines the standardized BP measurement protocol and rigorous QC program in KNHANES, aiming to ensure accurate and reliable BP data for epidemiological research and public health policymaking in South Korea. Graphical Abstract

Abstract Nocturnal blood pressure (BP) has been shown to have a significant predictive value for cardiovascular disease. In some cases, it has a superior predictive value for future cardiovascular outcomes than daytime BP. As efficacy of BP medications wanes during nighttime and early morning, control of nocturnal hypertension and morning hypertension can be difficult. As such, chronotherapy, the dosing of BP medication in the evening, has been an ongoing topic of interest in the field of hypertension. Some studies have shown that chronotherapy is effective in reducing nocturnal BP, improving non dipping and rising patterns to dipping patterns, and improving cardiovascular prognosis. However, criticism and concerns have been raised regarding the design of these studies, such as the Hygia study, and the implausible clinical benefits in cardiovascular outcomes considering the degree of BP lowering from bedtime dosing. Studies have shown that there is no consistent evidence to suggest that routine administration of antihypertensive medications at bedtime can improve nocturnal BP and early morning BP control. However, in some cases of uncontrolled nocturnal hypertension and morning hypertension, such as in those with diabetes mellitus, chronic kidney disease, and obstructive sleep apnea, bedtime dosing has shown efficacy in reducing evening and early morning BP. The recently published the Treatment in Morning versus Evening (TIME) study failed to demonstrate benefit of bedtime dosing in reducing cardiovascular outcomes in patients with hypertension. With issues of the Hygia study and negative results from the TIME study, it is unclear at this time whether routine bedtime dosing is beneficial for reducing cardiovascular outcomes.

Abstract Background Non-dipping or reverse dipping patterns are known to be associated with adverse cardiovascular prognosis among the general population and clinical cohort. Few large sized studies have explored factors including sleep duration and sleep quality related to nighttime blood pressure (BP) and nocturnal dipping patterns. Methods Among 5,360 patients enrolled in Korean multicenter nationwide prospective Registry of ambulatory BP monitoring (KORABP), 981 subjects with complete data on sleep duration, sleep quality assessed using a 4-point Likert scale, and clinical variables were included in the analysis. Phenotypes of nighttime BP pattern were categorized as extreme dipper, dipper, non-dipper, and reverse dipper. Hypertension was defined as a 24-h ambulatory BPs were 130/80 mmHg or higher. Results Among 981 subjects, 221 were normotensive, 359 were untreated hypertensive, and 401 were treated hypertensive. Age of the participants were 53.87 ± 14.02 years and 47.1% were female. In overall patients, sleep duration was 431.99 ± 107.61 min, and one to four points of sleep quality were observed in 15.5%, 30.0%, 30.4%, and 24.2%, respectively. Of the 760 hypertensive patients, extreme dipper, dipper, non-dipper, and reverse dipper were observed in 58 (7.63%), 277 (36.45%), 325 (42.76%), and 100 (13.16%), respectively. In multiple linear regression analysis, sleep duration (β = 0.0105, p

Purpose: This study aimed to investigate clinical practices and factors related to the outcomes of T-DM1 use in patients with HER2-positive metastatic breast cancer (mBC). Methods: We included patients with HER2-positive mBC who received T-DM1 as a palliative therapy between August 2017 and December 2018. The safety and outcomes of T-DM1, including overall response rate (ORR), progression-free survival (PFS), and overall survival (OS), were evaluated. A Cox proportional hazards model was used to estimate the hazard ratio and 95% confidence interval (CI) for mortality or progression to HER2-positive mBC. Results: In total, 824 patients were enrolled during the study period. The mean age of patients was 58 years, and 516 (62.6%) patients relapsed after curative treatment. Excluding a history of endocrine therapy, 341 (41.4%) patients previously received none or first-line chemotherapy, 179 (21.7%) received second-line therapy, and 303 (36.9%) received third-or later-line chemotherapy before T-DM1 therapy. During a median follow-up of 16.8 months, the ORR was 35%, the median PFS was 6.6 months, and the median OS was not reached. The clinical factors associated with the hazard of progression were age (

Financial difficulties experienced by cancer patients negatively impact the mental health of the patients. The objective of this study was to examine the mediating role of financial difficulties between physical symptoms and depression in patients with advanced cancer. A prospective, cross-sectional design was adopted in the study. The data were collected from 861 participants with advanced cancer in 15 different tertiary hospitals in Spain. The participants’ socio-demographic characteristics were collected using a standardized self-report form. Hierarchical linear regression models were used to explore the mediating role of financial difficulties. In the results, 24% of patients reported a high level of financial difficulties. Physical symptoms were positively associated with financial difficulties and depression (β = 0.46 and β = 0.43, respectively), and financial difficulties was positively associated with depression (β = 0.26). Additionally, financial difficulties played a role in explaining the relationship between physical symptoms and depression, showing a standardized regression coefficient of 0.43 which decreased to 0.39 after the financial difficulties were controlled. Healthcare professionals should consider the importance of providing financial resources and emotional support to help patients and their families cope with the financial burden associated with cancer treatment and its symptoms.

Abstract Background The balance of stroke risk reduction and potential bleeding risk associated with antithrombotic treatment (ATT) remains unclear in atrial fibrillation (AF) at non‐gender CHA2DS2‐VASc scores 0–1. A net clinical benefit (NCB) analysis of ATT may guide stroke prevention strategies in AF with non‐gender CHA2DS2‐VASc scores 0–1. Methods This multi‐center cohort study evaluated the clinical outcomes of treatment with a single antiplatelet (SAPT), vitamin K antagonist (VKA), and non‐VKA oral anticoagulant (NOAC) in non‐gender CHA2DS2‐VASc score 0–1 and further stratified by biomarker‐based ABCD score (Age [≥60 years], B‐type natriuretic peptide [BNP] or N‐terminal pro‐BNP [≥300 pg/mL], creatinine clearance [

Abstract Hypertension is the leading cause of death in human being, which shows high prevalence and associated complications that increase the mortality and morbidity. Controlling blood pressure (BP) is very important because it is well known that lowering high BP effectively improves patients’ prognosis. This review aims to provide a focused update of the 2018 Korean Hypertension Society Guidelines for the management of hypertension. The importance of ambulatory BP and home BP monitoring was further emphasized not only for the diagnosis but also for treatment target. By adopting corresponding BPs, the updated guideline recommended out-of-office BP targets for both standard and intensive treatment. Based on the consensus on corresponding BPs and Systolic Blood Pressure Intervention Trial (SPRINT) revisit, the updated guidelines recommended target BP in high-risk patients below 130/80 mmHg and it applies to hypertensive patients with three or more additional cardiovascular risk factors, one or more risk factors with diabetes, or hypertensive patients with subclinical organ damages, coronary or vascular diseases, heart failure, chronic kidney disease with proteinuria, and cerebral lacunar infarction. Cerebral infarction and chronic kidney disease are also high-risk factors for cardiovascular disease. However, due to lack of evidence, the target BP was generally determined at

The tribe Hydrangeeae displays a unique, distinctive disjunct distribution encompassing East Asia, North America and Hawaii. Despite its complex trait variations and polyphyletic nature, comprehensive phylogenomic and biogeographical studies on this tribe have been lacking. To address this gap, we sequenced and characterized 28 plastomes of Hydrangeeae. Our study highlights the highly conserved nature of Hydrangeaceae chloroplast (cp) genomes in terms of gene content and arrangement. Notably, synapomorphic characteristics of tandem repeats in the conserved domain of accD were observed in the Macrophyllae, Chinenses, and Dichroa sections within the Hydrangeeae tribe. Additionally, we found lower expression of accD in these sections using structure prediction and quantitative real-time PCR analysis. Phylogenomic analyses revealed the subdivision of the Hydrangeeae tribe into two clades with robust support values. Consistent with polyphyletic relationships, sect. Broussaisia was identified as the basal group in the tribe Hydrangeeae. Our study also provides insights into the phylogenetic relationships of Hydrangea petiolaris in the Jeju and Ulleung Island populations, suggesting the need for further studies with more samples and molecular data. Divergence time estimation and biogeographical analyses suggested that the common ancestors of the tribe Hydrangeeae likely originated from North America and East Asia during the Paleocene period via the Bering Land Bridge, potentially facilitating migration within the tribe between these regions. In conclusion, this study enhances our understanding of the evolutionary history and biogeography of the tribe Hydrangeeae, shedding light on the dispersal patterns and origins of this intriguing plant group with its unique disjunct distribution.

Abstract Home blood pressure measurement (HBPM) has the advantage of measuring blood pressure (BP) multiple times over a long period. HBPM effectively diagnoses stress-induced transient BP elevations (i.e., white coat hypertension), insufficient BP control throughout the day (i.e., masked hypertension), and even BP variability. In most cases, HBPM may increase self-awareness of BP, increasing the compliance of treatment. Cumulative evidence has reported better improved predictive values of HBPM in cardiovascular morbidity and mortality than office BP monitoring. In this position paper, the Korean Society of Hypertension Home Blood Pressure Forum provides comprehensive information and clinical importance on HBPM.

Background Despite recent advances in first-line chemotherapy for advanced pancreatic cancer, standard treatment after the failure of initial chemotherapy has not been established. Hence, we aimed to retrospectively analyze the clinical characteristics and outcomes of second-line chemotherapy in patients with advanced pancreatic cancer. Methods We reviewed the clinical data of patients with advanced pancreatic cancer who underwent palliative chemotherapy at Kosin University Gospel Hospital between January 2013 and October 2020. Results Among 366 patients with advanced pancreatic cancer who had received palliative chemotherapy, 104 (28.4%) underwent at least one cycle of second-line chemotherapy. The median age of the patients at the time of initiating second-line treatment was 62 years (interquartile range, 57–62 years), and 58.7% (61 patients) of them were male. The common second-line chemotherapy regimens were 5-fluorouracil (FU) plus leucovorin, irinotecan, and oxaliplatin (33 patients, 31.7%); gemcitabine/nab-paclitaxel (29, 27.9%), gemcitabine±erlotinib (13, 12.5%); and oxaliplatin and 5-FU/leucovorin (12, 11.5%). The median overall survival (OS) and progression-free survival were 6.4 months (95% confidence interval [CI], 4.5–8.6 months) and 4.5 months (95% CI, 2.7–6.3 months), respectively. In a multivariate analysis, poor performance status (PS) (hazard ratio [HR], 2.247; p=0.021), metastatic disease (HR, 2.745; p=0.011), and elevated carcinoembryonic antigen (CEA) levels (HR, 1.939; p=0.030) at the beginning of second-line chemotherapy were associated with poor OS. Conclusion The survival outcome of second-line chemotherapy for advanced pancreatic cancer remains poor. However, PS, disease extent (locally advanced or metastatic), and CEA level may help determine patients who could benefit from second-line treatment.

Purpose A community program is an efficient model for improving the management of chronic diseases such as hypertension, diabetes, and dyslipidemia. A specific blood pressure (BP) measurement protocol was developed for community settings in which BP was measured by the interviewer at the interviewee’s home. Materials and methods In the 2018 Korean Community Health Survey, BP was measured twice at a five-minute interval after a five-minute resting period at the beginning of the survey. In 2019, BP was measured at the end of the survey after a two-minute rest and was obtained as three measurements at one-minute intervals. As factors related to BP level, stressful stimuli within 30 min before BP measurement such as smoking, caffeine, and/or exercise; duration of rest; and survey year were analysed. Results The mean age of participants was 55.2 years, and females accounted for 55.4% of the participants (n = 399,838). Stressful stimuli were observed in 21.9% of the participants in 2018 (n = 188,440) and 11.3% in 2019 (n = 211,398). Duration of rest was 0 min (2.1%), two minutes (55.0%), and five minutes (47.9%). When adjusted for age, sex, body mass index, antihypertensive medication, the arm of measurement, survey year (beta= −4.092), stressful stimuli (beta = 0.834), and resting time (beta = −1.296 per one minute of rest) were significant factors for mean systolic BP. A two-minute rest was not a significant factor in mean BP. The differences in adjusted mean systolic BPs were significant for rest times of five minutes vs. two minutes (3.1 mmHg, p

Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe cutaneous adverse drug reaction; reported cases are sometimes imatinib mesylate induced. The main treatment is the withdrawal of the causative drug, and most cases with imatinib-induced DRESS syndrome required withdrawal of imatinib. However, in such cases involving anticancer drugs, this may compromise cancer treatment. Herein, we report a patient with imatinib-induced DRESS syndrome that was successfully treated with reslizumab while continuing imatinib treatment. A 65-year-old female presented with facial edema and generalized skin rash after being given 400 mg imatinib 2 weeks ago for metastatic gastrointestinal stromal tumor. After stopping imatinib, the clinical symptoms improved. Imatinib desensitization was performed, and it was administered again. However, the clinical symptoms reappeared more severely 2 months after restart of imatinib, and the peripheral absolute eosinophil count increased to 1,690/μL. A diagnosis of imatinib-induced DRESS syndrome was made, based on the Registry of Severe Cutaneous Adverse Reactions (RegiSCAR) criteria. Imatinib desensitization was repeated, but the clinical symptoms reappeared, and the peripheral eosinophilia persisted. We administered reslizumab, an interleukin-5 monoclonal antibody, without cessation of imatinib. The absolute eosinophil count decreased immediately, and the clinical symptoms improved gradually. After 2 weeks, the clinical symptoms reappeared mildly, but after administering reslizumab again, these disappeared completely. Reslizumab can be considered in the management of DRESS syndrome in cases wherein the causative medication needs to be continued.

Background/Aims The clinical characteristics of patients with masked uncontrolled hypertension (MUCH) have been poorly defined, and few studies have investigated the clinical predictors of MUCH. We investigated the demographic, clinical, and blood pressure (BP) characteristics of patients with MUCH and proposed a prediction model for MUCH in patients with hypertension. Methods We analyzed 1,986 subjects who were enrolled in the Korean Ambulatory Blood Pressure Monitoring (Kor-ABP) Registry and taking antihypertensive drugs, and classified them into the controlled hypertension (n = 465) and MUCH (n = 389) groups. MUCH was defined as the presence of a 24-hour ambulatory mean systolic BP ≥ 130 mmHg and/or diastolic BP ≥ 80 mmHg in patients treated with antihypertensive drugs, having normal office BP. Results Patients in the MUCH group had significantly worse metabolic profiles and higher office BP, and took significantly fewer antihypertensive drugs compared to those in the controlled hypertension group. Multivariate logistic regression analyses identified high office systolic BP and diastolic BP, prior stroke, dyslipidemia, left ventricular hypertrophy (LVH, ≥ 116 g/m2 for men, and ≥ 96 g/m2 for women), high heart rate (≥ 75 beats/min), and single antihypertensive drug use as independent predictors of MUCH. A prediction model using these predictors showed a high diagnostic accuracy (C-index of 0.839) and goodness-of-fit for the presence of MUCH. Conclusions MUCH is associated with a high-normal increase in office BP and underuse of antihypertensive drugs, as well as dyslipidemia, prior stroke, and LVH, which could underscore achieving optimal BP control. The proposed model accurately predicts MUCH in patients with controlled office BP.

Summary: Here, we present a protocol for constructing direct electron transfer (DET)-based enzyme-electrodes using gold-binding peptide (GBP). We describe fusion of four GBPs to flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase gamma-alpha complex (GDHγα), as model oxidoreductase, to generate four GDHγα variants. We then detail the measurements of catalytic and bioelectrochemical properties of these GDHγα variants on electrode together with surface morphology of GDHγα variants immobilized on gold surface. This protocol is useful for construction and validation of enzyme-based electrocatalytic system.For complete details on the use and execution of this protocol, please refer to Lee et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary Most extremophilic anaerobes possess a sulfur formation (Suf) system for Fe–S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe–S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW‐1. Comparative genomics of the order Thermotogales revealed that the feather‐degrading F. islandicum AW‐1 has a complete Suf‐like machinery (SufCBDSU) that is highly expressed in cells grown on native feathers in the absence of elemental sulfur (S0). On the other hand, F. islandicum AW‐1 exhibited a significant retardation in the Suf system‐mediated keratin degradation in the presence of S0. Detailed differential expression analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Indeed, addition of SufS–SufU to cell extracts containing keratinolytic proteases accelerated keratin decomposition in vitro under reducing conditions. Remarkably, mass spectrometric analysis of extracellular and intracellular levels of amino acids suggested that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf‐like machinery including the SufS–SufU complex may contribute to sulfur availability for an extracellular reducing environment as well as intracellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions.

Abstract Background The necessity to develop high lipid-producing microalgae is emphasized for the commercialization of microalgal biomass, which is environmentally friendly and sustainable. Nannochloropsis are one of the best industrial microalgae and have been widely studied for their lipids, including high-value polyunsaturated fatty acids (PUFAs). Many reports on the genetic and biological engineering of Nannochloropsis to improve their growth and lipid contents have been published. Results We performed insertional mutagenesis in Nannochloropsis salina, and screened mutants with high lipid contents using fluorescence-activated cell sorting (FACS). We isolated a mutant, Mut68, which showed improved growth and a concomitant increase in lipid contents. Mut68 exhibited 53% faster growth rate and 34% higher fatty acid methyl ester (FAME) contents after incubation for 8 days, resulting in a 75% increase in FAME productivity compared to that in the wild type (WT). By sequencing the whole genome, we identified the disrupted gene in Mut68 that encoded trehalose-6-phosphate (T6P) synthase (TPS). TPS is composed of two domains: TPS domain and T6P phosphatase (TPP) domain, which catalyze the initial formation of T6P and dephosphorylation to trehalose, respectively. Mut68 was disrupted at the TPP domain in the C-terminal half, which was confirmed by metabolic analyses revealing a great reduction in the trehalose content in Mut68. Consistent with the unaffected N-terminal TPS domain, Mut68 showed moderate increase in T6P that is known for regulation of sugar metabolism, growth, and lipid biosynthesis. Interestingly, the metabolic analyses also revealed a significant increase in stress-related amino acids, including proline and glutamine, which may further contribute to the Mut68 phenotypes. Conclusion We have successfully isolated an insertional mutant showing improved growth and lipid production. Moreover, we identified the disrupted gene encoding TPS. Consistent with the disrupted TPP domain, metabolic analyses revealed a moderate increase in T6P and greatly reduced trehalose. Herein, we provide an excellent proof of concept that the selection of insertional mutations via FACS can be employed for the isolation of mutants with improved growth and lipid production. In addition, trehalose and genes encoding TPS will provide novel targets for chemical and genetic engineering, in other microalgae and organisms as well as Nannochloropsis.

Circulating tumor cells (CTCs) have emerged as liquid biopsy biomarker providing non-invasive assessment of cancer progression and biology. We investigated whether longitudinal analysis of CTCs could monitor disease progression, response to chemotherapy, and survival in patients with unresectable pancreatic ductal adenocarcinoma (PDAC). A total of 52 patients with PDAC were prospectively enrolled in this study. Peripheral blood samples were serially collected at the time of diagnosis and after chemotherapy with clinical assessments. CTCs were isolated through a centrifugal microfluidic disc, enumerated with immunostaining against Epithelial cell adhesion molecule (EpCAM), Cytokeratin (CK), Plectin-1 and CD45, and identified by an automated imaging system. One or more CTCs were detected in 84.62% patients with unresectable PDAC at the time of diagnosis. CTC numbers were not statistically different across tumor sizes, location and metastatic sites. The absolute number of CTCs after chemotherapy was inversely related to overall survival (OS), and the decreased number of CTCs after chemotherapy was significantly associated with longer OS in patients with PDAC. Identifying CTCs and monitoring CTC changes after chemotherapy could be a useful prognostic marker for survival in patients with unresectable PDACs.

Summary: Oriented enzyme immobilization on electrodes is crucial for interfacial electrical coupling of direct electron transfer (DET)-based enzyme-electrode systems. As inorganic-binding peptides are introduced as molecular binders and enzyme-orienting agents, inorganic-binding peptide-fused enzymes should be designed and constructed to achieve efficient DET. In this study, it is aimed to compare the effects of various gold-binding peptides (GBPs) fused to enzymes on electrocatalytic activity, bioactivity, and material-binding behaviors. Here, GBPs with identical gold-binding properties but different amino acid sequences were fused to the FAD-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) to generate four GDHγα variants. The structural, biochemical, mechanical, and bioelectrochemical properties of these GDHγα variants immobilized on electrode were determined by their fused GBPs. Our results confirmed that the GBP type is vital in the design, construction, and optimization of GBP-fused enzyme-modified electrodes for facile interfacial DET and practical DET-based enzyme-electrode systems.

Abstract DNA transfer between internal organelles such as the nucleus, mitochondrion, and plastid is a well-known phenomenon in plant evolution, and DNA transfer from the plastid and mitochondrion to the nucleus, from the plastid to the mitochondrion, and from the nucleus to the mitochondrion has been well-documented in angiosperms. However, evidence of the transfer of mitochondrial DNA (mtDNA) to the plastid has only been found in three dicotyledons and one monocotyledon. In the present study, we characterised and analysed two chloroplast (cp) genome sequences of Convallaria keiskei and Liriope spicata, and found that C. keiskei has the largest cp genome (162,109 bp) in the Asparagaceae. Interestingly, C. keiskei had a ~3.3-kb segment of mtDNA in its cp genome and showed similarity with the mt gene rpl10 as a pseudogene. Further analyses revealed that mtDNA transfer only occurred in C. keiskei in the Nolinoideae, which diverged very recently (7.68 million years ago (mya); 95% highest posterior density (HPD): 14.55–2.97 mya). These findings indicate that the C. keiskei cp genome is unique amongst monocotyledon land plants, but further work is necessary to understand the direction and mechanism involved in the uptake of mtDNA by the plastid genome of C. keiskei.

Abnormal accumulation of misfolded tau aggregates is a pathological hallmark of various tauopathies including Alzheimer’s disease (AD). Although tau is a cytosolic microtubule-associated protein enriched in neurons, it is also found in extracellular milieu, such as interstitial fluid, cerebrospinal fluid, and blood. Accumulating evidence showed that pathological tau spreads along anatomically connected areas in the brain through intercellular transmission and templated misfolding, thereby inducing neurodegeneration and cognitive dysfunction. In line with this, the spatiotemporal spreading of tau pathology is closely correlated with cognitive decline in AD patients. Although the secretion and uptake of tau involve multiple different pathways depending on tau species and cell types, a growing body of evidence suggested that tau is largely secreted in a vesicle-free forms. In this regard, the interaction of vesicle-free tau with membrane is gaining growing attention due to its importance for both of tau secretion and uptake as well as aggregation. Here, we review the recent literature on the mechanisms of the tau-membrane interaction and highlights the roles of lipids and proteins at the membrane in the tau-membrane interaction as well as tau aggregation.

Circulating tumor cells (CTCs) are useful biomarkers of many solid tumors, but are infrequently detected in early stage pancreatic ductal adenocarcinomas (PDACs). The first drainage of pancreatic venous blood flow come to portal vein and pass through the liver, and they finally go out for peripheral blood. We thought that comparing CTCs from portal vein and peripheral blood could enable us to understand the clinical meaning of CTCs from each different site in PDACs. Therefore, we aimed to determine 1) whether CTCs could be reliably identified in early stages (operable) of PDACs, 2) if there are any differences in the detected number of CTC in portal vein blood and peripheral blood, and 3) whether CTCs can be sensitive biomarkers for the prognosis of resectable PDAC patients. Newly diagnosed PDAC patients who underwent operation with curative intention between 2013 and 2015 were prospectively enrolled. Blood draws from portal and peripheral vein ran through the microfabricated porous filter, and anti-epithelial cell adhesion molecule (EpCAM) and anti-Plectin-1 antibodies were used for CTC identification. Baseline clinical characteristics, tumor characteristics, treatment, and clinical outcomes were assessed. The clinical stages of the 32 enrolled patients were as follows: IA/IB 1 (3.1%); IIA 9 (28.1%); IIB 17 (53.1%); III 5 (15.6%). Twenty-seven patients (84.4%) received R0 resection, while five patients (15.6%) received R1 resection. EpCAM+ CTCs were detected in 20 portal blood (62.5%) and 22 peripheral blood (68.8%). Plectin-1+ CTCs were identified in 14 portal blood (43.8%) and 16 peripheral blood (50%). Plectin-1-expressing CTCs were picked from CTC platform (microfabricated porous filter) and we could find out all KRAS mutation. Patients with detectable EpCAM+ CTC less than one in peripheral blood showed longer overall survival (OS) compared to patients with detectable CTCs more than one (35.5 months vs. 16.0 months). EpCAM and Plectin-1 successfully identified CTCs at the early stage of PDACs. Also, the number of CTCs could be a prognostic marker for survival in resectable PDACs.

Objectives: Osteolytic bone lesions are common complications in multiple myeloma (MM), and can have an impact on quality of life due to the risk of fractures. Trabecular bone score (TBS) is a novel texture index derived from dual energy x-ray absorptiometry (DXA) of lumbar spine (LS) images that provides information about bone microarchitecture. The aim of this study was to evaluate whether TBS is useful in predicting bone fractures in MM patients. Methods: TBS was calculated retrospectively from existing DXA images of the LS, in 20 patients with newly diagnosed MM. We analyzed the development of fractures in these patients. Results: The median age of the patients was 66 years (range, 49–77 years). Osteolytic bone lesions were observed in 18 patients (90%) at the time of diagnosis. The median duration of follow-up was 40.0 months (95% confidence interval [CI], 33.2–46.2), 6 fracture events (long-bone fractures in 5 events, vertebral fracture in 1) occurred in 5 patients (25%). There were no significant differences between patients who experienced new onset fractures and patients who did not for all TBSs and T-scores, although the fracture group had lower levels than the no fracture group. However, among TBSs of individual LSs, only L2 showed significantly lower scores in patients who developed fractures (1.135 ± 0.085 [95% CI, 1.030–1.241] vs. 1.243 ± 0.169 [95% CI, 1.149–1.336], P = 0.032). Conclusions: TBS of the LS in MM patients may be helpful in predicting development of fractures; however, further investigation is needed. Keywords: Multiple myeloma, Trabecular bone score, Fractures

Gelsolin is an actin binding protein present in blood plasma and in cytoplasm of cells including macrophages. Gelsolin has important functions in cell cycle regulation, apoptotic regulation, and morphogenesis. Even though bone marrow macrophages and serum factors are critical for regulating erythropoiesis, the role of gelsolin on human erythroblasts has not been studied. Here, we investigated the effects of human recombinant plasma gelsolin (pGSN) on human immature erythroblasts. CD34+ cells isolated from cord blood were differentiated into erythroid cells in serum-free medium. When pGSN was applied to the culture medium, it accelerated basophilic and polychromatic erythroblast maturation and increased the enucleation rate with highly expressed erythropoiesis-related mRNAs. Also, pGSN was effective in reducing dysplastic changes caused by vincristine, suggesting its role in cell cycle progression at G2/M checkpoints. Also, pGSN activated caspase-3 during maturation stages in which caspase-3 functions as a non-apoptotic maturational signal or a pro-apoptotic signal depending on maturation stages. Our results suggest that pGSN has a pivotal role in maturation of erythroblasts and this factor might be one of the way how bone marrow macrophages and previously unknown serum factors work to control erythropoiesis. pGSN might be used as additive for in vitro production of erythrocytes. Keywords: Gelsolin, Erythropoiesis, Dysplasia, Red blood cells

Acute humoral xenograft rejection (AHXR), characterized by thrombin generation and endothelial cell activation, should be overcome for the success of xenotransplantation. Fibrinogen-like protein 2 (fgl2) expressed on endothelial cells can convert prothrombin to thrombin directly, which indicates that the induced fgl2 expression in activated endothelial cells can contribute to thrombosis. In xenotransplant condition, the interaction between human CD40L and porcine endothelial CD40 can activate endothelial cells. In this study, we investigated the effect of endothelial cell activation through the interaction between human CD40L and porcine CD40 on fgl2 expression and its function as a direct prothrombinase. We found that CD40 stimulation up-regulated fgl2 expression as well as its enzymatic activity in porcine endothelial cells. Moreover, functional studies using knock-down system showed that the major factor converting human prothrombin to thrombin is fgl2 protein expressed on porcine endothelial cells. Overall, this study demonstrates that fgl2 expression can be induced by xenogeneic CD40 signal on endothelial cells and contribute to thrombin generation.

Social networking service(SNS) helps users manage, share and delivery a vast information as a communication tool. When users read crisis news in SNS, they communicate the information with others by considering not only their belief (i.e., cognitive risk perception) but also emotion (i.e., affective risk perception). However, few researches have been interested in the construct of communication behaviors of crisis in SNS. This study aimed to explore the role of risk perception (cognitive and affective risk perception) between crisis responsibility and communication behavior through dual processing theory. As a result of the empirical analysis, crisis responsibility had a positive effect on cognitive risk perception and affective risk perception. In addition, cognitive risk perception had no significant effect on communication behavior whereas affective risk perception had influence on communication behavior positively. Thus, our findings may predict that the affective risk perception through crisis responsibility is more potentially important to communication behavior such as sharing information rather than cognitive risk perception. The results give insightful ideas why marketer should reduce perceived emotion caused by risk to strengthen prospective SNS users understanding of communication behavior intention.

ObjectivesThe addition of bevacizumab to standard chemotherapy has been improved survival outcomes in patients with metastatic colorectal cancer. However, the combination of bevacizumab with oxaliplatin-based chemotherapy as first-line treatment showed limited survival benefit. The purpose of this study was to investigate the clinical efficacy and toxicity of the combination of bevacizumab to oxaliplatin and leucovorin (FOLFOX4) in the first-line treatment of patient with metastatic colorectal cancer. MethodsBetween December 2004 and September 2009, medical records of patients who were diagnosed with metastatic colorectal cancer and received the first line chemotherapy with bevacizumab and FOLFOX4, were retrospectively reviewed. ResultsA total of forty patients were analyzed. The median age of the patients was 55 years (range, 33-80), and 55% was male. The patients received a total of 206 cycles of therapy (median 4 cycles per patient; range 1 – 15 cycles). Of these 40 patients, none achieved complete response (CR) and 15 achieved a partial response (PR), for the overall response rate (ORR) 37.5% (95% CI, 22.5-52.5). Median progression free survival (PFS) was 6.9 months (95% CI, 3.4-10.5) and median overall survival (OS) was 22.6 months (95% CI, 17.3-27.8The most common grade 3 or 4 hematologic toxicity and non-hematologic toxicity were neutropenia (10.0%) and diarrhea (10.0%), respectively. Two patients experienced gastrointestinal perforation. ConclusionsIn this study, the combination bevacizumab with FOLFOX4 was associated with favorable OS, but did not showed favorable PFS and ORR.

Objective Burial is associated with environmental effects such as the contamination of ground or surface water with biological materials generated during the decomposition process. Therefore, bacterial communities in leachates originating from the decomposing bovine carcasses were investigated. Methods To understand the process of bovine (Hanwoo) carcass decomposition, we simulated burial using a lab-scale reactor with a volume of 5.15 m3. Leachate samples from 3 carcasses were collected using a peristaltic pump once a month for a period of 5 months, and bacterial communities in samples were identified by pyrosequencing of the 16S rRNA gene. Results We obtained a total of 110,442 reads from the triplicate samples of various sampling time points (total of 15 samples), and found that the phylum Firmicutes was dominant at most sampling times. Differences in the bacterial communities at the various time points were observed among the triplicate samples. The bacterial communities sampled at 4 months showed the most different compositions. The genera Pseudomonas and Psychrobacter in the phylum Proteobacteria were dominant in all of the samples obtained after 3 months. Bacillaceae, Clostridium, and Clostridiales were found to be predominant after 4 months in the leachate from one carcass, whereas Planococcaceae was found to be a dominant in samples obtained at the first and second months from the other two carcasses. The results showed that potentially pathogenic microbes such as Clostridium derived from bovine leachate could dominate the soil environment of a burial site. Conclusion Our results indicated that the composition of bacterial communities in leachates of a decomposing bovine shifted continuously during the experimental period, with significant changes detected after 4 months of burial.

The cystic cavity that develops following injuries to brain or spinal cord is a major obstacle. Here the authors show an injection of imidazole poly(organophosphazenes), a hydrogel with thermosensitive sol–gel transition behavior, almost completely eliminates cystic cavities in a clinically relevant rat spinal cord injury model.

Background Stroke and thromboembolic events may still occur in “clinically low‐risk” atrial fibrillation (AF) patients as categorized by CHA2DS2‐VASc score. Our aim was to assess the proportion of “clinically low‐risk” patients using a nongender CHA2DS2‐VASc (ie, CHA2DS2‐VA) score of 0 to 1 among patients who experienced AF‐associated stroke and to identify markers associated with stroke in “clinically low‐risk” patients. Methods and Results We retrospectively recruited nonvalvular AF patients who experienced embolic stroke between 2013 and 2016 from 9 institutes in Korea. AF patients with CHA2DS2‐VA score of 0 to 1 at the time of stroke were analyzed and compared with “clinically low‐risk” AF patients without stroke. A total of 3033 subjects with AF‐associated stroke were recruited. Of these, 583 patients (19.2%) had CHA2DS2‐VA score of 0 to 1. On multivariate analysis, age (≥60 years), N‐terminal pro B‐type natriuretic peptide (≥300 pg/mL), creatinine clearance (

The precise determination of the chemical composition in crops is important to identify their nutritional and functional value. The current study performed a systematic delineation of the rice metabolome, an important staple in Asia, to investigate the following: (1) comparative features between brown and white rice; (2) variety-specific composition (Ilpum vs. Odae); and (3) cultivation of region-dependent metabolic content. Global metabolic profiling and data-driven statistics identified the exclusive enrichment of compounds in brown rice compared to white rice. Next, the authors investigated a variety-governed metabolic phenotype among various geo-environmental factors. Odae, the early-ripening cultivar, showed higher contents of most chemicals compared to the late-ripening cultivar, Ilpum. The authors identified regional specificity for cultivation among five areas in Korea which were characterized by polishing degree and cultivar type. Finally, the current study proposes a possible linkage of the region-specific metabolic signatures to soil texture and total rainfall. In addition, we found tryptophan metabolites that implied the potential for microbe–host interactions that may influence crop metabolites.

Abstract Objectives With the rapid increase in the prevalence of diabetes, the age groups of diabetic patients are becoming diversified. This study will examine the degree of obesity, insulin resistance, and insulin secretion ability among patients first diagnosed with diabetes according to age and gender. Methods The subjects of this study included 616 patients who were first diagnosed with diabetes during a routine physical examination. This sample was obtained from a total of 28,075 adults aged 19 years and older who received the examination among 33,829 participants in the Korea National Health & Nutrition Examination Survey (KNHANES) from 2007–2010. The subjects were categorized by age into young age (age: 19 – 39 years), middle age (age: 40 – 59 years), and old age (age: 60 years and older). The degree of obesity was categorized according to body mass index (BMI) into normal weight (BMI: 18.5 ∼ 22.9), overweight (BMI: 23 ∼ 24.9), and obesity (BMI: 25 or above). Insulin resistance was evaluated by homeostatic model assessment of insulin resistance (HOMA-IR). Results It was found that 14.1% (n = 87) of a total of 616 subjects (324 men, 292 women) were in the young age group, 43.8% (n = 270) were in the middle age group, and 42.1% (n = 259) were in the old age group. In addition, 83.3% of men that were overweight or obesity were in the young age group, while 79.2% and 60.5% were in the middle age and old age groups, respectively. A total of 82.2% of women that were overweight or obesity were in the young age group, while 79.5% and 77% were in the middle age and old age groups, respectively. For men, the more obesity they were in all age groups, the higher their HOMA-IR. For women, the more obesity they were in the young age and middle age groups, the higher their HOMA-IR; however, women in the old age group showed the highest HOMA-IR when they were of normal weight. Conclusion Among diabetic patients first diagnosed with the disease in Korea, the youth population had the highest obesity rate. Insulin resistance increases as an individual's weight increases among those patients who are first diagnosed with diabetes; the only exception noted is for elderly women.

Abstract Objectives The both values of neutrophil lymphocyte ratio (NLR) and platelet lymphocyte ratio (PLR) were reported as indexes of systemic inflammation and readily available and inexpensive prognostic markers in patients with solid cancer. The objective of this study was to clarify whether the NLR and PLR were significant prognostic markers in Korean diffuse large B-cell lymphoma (DLBCL) patients treated with R-CHOP as a first line therapy. Methods : We retrospectively collected the clinical data of ninety nine DLBCL patients treated with R-CHOP from 2004 to 2012 and analyzed. NLR and PLR were calculated from complete blood count (CBC) and differential leukocyte count. Results : In univariate analysis, NLR was significantly associated with 5-year progression free survival(PFS) rate (P= 0.039), but not significantly associated with 5-year overal survival (OS) rate (P= 0.276). PLR was not significantly associated with 5-year PFS (P= 0.632) and OS rate (P= 0.855). In multivariate analysis, NLR was not a significant independent prognostic factor for 5-year PFS (P= 0.415) and OS rate (P= 0.991). Conclusion : The NLR can be considered a useful predictor of survival outcome. The PLR is not a valid predictor of survival outcome.

The complete chloroplast genome sequence of Maianthemum dilatatum is sequenced and analyzed. The chloroplast genome is 156,921 bp, with 36.7% GC content. A pair of inverted repeats of 26,468 bp is separated by a large single-copy region (85,554 bp) and a small single-copy region (18,431 bp). It encodes 86 protein-coding genes, 38 tRNA genes and 8 rRNA genes. Of 132 individual genes, 19 genes are duplicated in the IR regions, while 14 genes are encoded with one intron and three genes with two introns.

Activity-guided fractionation of a methanolic extract of the leaves of Eremochloa ophiuroides (centipede grass) using a pancreatic lipase inhibitory assay led to the isolation and identification of a new C-glycosidic flavone, luteolin 6-C-b-D-boivinopyranoside (1), as well as eight known compounds. The structures of these compounds were established on the basis of interpretation of their spectroscopic data. Among these isolates, the C-glycosidic flavones 1–5 showed potent inhibitory effects on pancreatic lipase, with IC50 values ranging from 18.5 ± 2.6 to 50.5 ± 3.9 mM.

High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium's minimal information (MIxS) and NCBI's BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.

In pig-to-primate xenotransplantation, multiple transgenic pigs are required to overcome a series of transplant rejections. The generation of multiple transgenic pigs either by breeding or the introduction of several mono-cistronic vectors has been hampered by the differential expression patterns of the target genes. To achieve simultaneous expression of multiple genes, a poly-cistronic expression system using the 2A peptide derived from the Thosea asigna virus (T2A) can be considered an alternative choice. Before applying T2A expression system to pig generation, the expression patterns of multiple genes in this system should be precisely evaluated. In this study, we constructed several bi-cistronic T2A expression vectors, which combine target genes that are frequently used in the xenotransplantation field, and introduced them into porcine fibroblasts. The proteins targeted to the same or different subcellular regions were efficiently expressed without affecting the localization or expression levels of the other protein. However, when a gene with low expression efficiency was inserted into the upstream region of the T2A sequences, the expression level of the downstream gene was significantly decreased compared with the expression efficiency without the insertion. A small interfering RNA targeting one gene in this system resulted in the significant downregulation of both the target gene and the other gene, indicating that multiple genes combined into a T2A expression vector can be considered as a single gene in terms of transcription and translation. In summary, the efficient expression of a downstream gene can be achieved if the expression of the upstream gene is efficient.

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.