Mammalian genome research resources available from the National BioResource Project in Japan.

Mammalian genome research has conventionally involved mice and rats as model organisms for humans. Given the recent advances in life science research, to understand complex and higher-order biological phenomena and to elucidate pathologies and develop therapies to promote human health and overcome diseases, it is necessary to utilize not only mice and rats but also other bioresources such as standardized genetic materials and appropriate cell lines in order to gain deeper molecular and cellular insights. The Japanese bioresource infrastructure program called the National BioResource Project (NBRP) systematically collects, preserves, controls the quality, and provides bioresources for use in life science research worldwide. In this review, based on information from a database of papers related to NBRP bioresources, we present the bioresources that have proved useful for mammalian genome research, including mice, rats, other animal resources; DNA-related materials; and human/animal cells and microbes.

T follicular helper cells have distinct modes of migration and molecular signatures in naive and memory immune responses.

B helper follicular T (Tfh) cells are critical for long-term humoral immunity. However, it remains unclear how these cells are recruited and contribute to secondary immune responses. Here we show that primary Tfh cells segregate into follicular mantle (FM) and germinal center (GC) subpopulations that display distinct gene expression signatures. Restriction of the primary Tfh cell subpopulation in the GC was mediated by downregulation of chemotactic receptor EBI2. Following collapse of the GC, memory T cells persisted in the outer follicle where they scanned CD169(+) subcapsular sinus macrophages. Reactivation and intrafollicular expansion of these follicular memory T cells in the subcapsular region was followed by their extrafollicular dissemination via the lymphatic flow. These data suggest that Tfh cells integrate their antigen-experience history to focus T cell help within the GC during primary responses but act rapidly to provide systemic T cell help after re-exposure to the antigen.

Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.

Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.

Verification of pharmacogenomics-based algorithms to predict warfarin maintenance dose using registered data of Japanese patients.

PURPOSE: Large inter-individual differences in warfarin maintenance dose are mostly due to the effect of genetic polymorphisms in multiple genes, including vitamin K epoxide reductase complex 1 (VKORC1), cytochromes P450 2C9 (CYP2C9), and cytochrome P450 4F2 (CYP4F2). Thus, several algorithms for predicting the warfarin dose based on pharmacogenomics data with clinical characteristics have been proposed. Although these algorithms consider these genetic polymorphisms, the formulas have different coefficient values that are critical in this context. In this study, we assessed the mutual validity among these algorithms by specifically considering racial differences. METHODS: Clinical data including actual warfarin dose (AWD) of 125 Japanese patients from our previous study (Eur J Clin Pharmacol 65(11):1097-1103, 2009) were used as registered data that provided patient characteristics, including age, sex, height, weight, and concomitant medications, as well as the genotypes of CYP2C9 and VKORC1. Genotyping for CYP4F2*3 was performed by the PCR method. Five algorithms that included these factors were selected from peer-reviewed articles. The selection covered four populations, Japanese, Chinese, Caucasian, and African-American, and the International Warfarin Pharmacogenetics Consortium (IWPC). RESULTS: For each algorithm, we calculated individual warfarin doses for 125 subjects and statistically evaluated its performance. The algorithm from the IWPC had the statistically highest correlation with the AWD. Importantly, the calculated warfarin dose (CWD) using the algorithm from African-Americans was less correlated with the AWD as compared to those using the other algorithms. The integration of CYP4F2 data into the algorithm did not improve the prediction accuracy. CONCLUSION: The racial difference is a critical factor for warfarin dose predictions based on pharmacogenomics.

Antibiotic-associated adverse drug events at a Japanese academic hospital.

INTRODUCTION: To promote antimicrobial stewardship activity, an understanding of the incidence of antibiotic-associated adverse drug events (ADEs) is essential. In this study, we aimed to describe the occurrence of antibiotic-associated ADEs at our hospital. METHODS: We retrospectively searched the ADE registration system in Osaka University Hospital between 2010 and 2017. Registrations of ADEs were dependent on the patients' drug history and clinical course after hospitalization. We classified the data according to types of ADEs (gastrointestinal, hepatobiliary, renal, cardiac, respiratory, hematologic, neurologic, dermatologic, and musculoskeletal) and antibiotic class. RESULTS: During the study period, we found 707 cases of antibiotic-associated ADEs, accounting for 22.3% of all the cases. Beta-lactam antibiotics constitute more than half of the cases (51.3%). The most common ADE was dermatologic abnormalities (53.4%), followed by liver dysfunction (9.7%) and gastrointestinal symptoms (8.9%). Among all antibiotics, oral third-generation cephalosporins were frequently reported as offending drugs (107 cases), accounting for 29.5% of beta-lactam ADEs and 46.3% of cephem ADEs. CONCLUSION: Antibiotic-associated ADEs covered approximately 20% of all the ADEs at our hospital. We believe that the data would be helpful in ensuring patient safety by promoting antimicrobial stewardship in hospitals.

Hemoglobin Value Is the Most Important Factor in the Development of Hand-Foot Syndrome under the Capecitabine Regimen.

BACKGROUND: Hand-foot syndrome (HFS) is a common side effect that has a high occurrence rate with capecitabine (Cape) chemotherapy. However, little is known about the risk factors of developing HFS under the Cape regimen. Our aim was to examine these risk factors. METHODS: A univariate analysis was used to determine the risk factors associated with developing HFS, and we calculated the effect sizes between the patients who developed HFS compared to those who did not. RESULTS: Of the 52 patients enrolled in our research, 24 (46.2%) developed HFS. This group was significantly associated with hemoglobin (Hb) values (p < 0.001), and the effect size (1.21) was more than moderate. The receiver operating characteristic curve analysis confirmed 12 mg/dl Hb as the best diagnostic cut-off value for developing HFS. The sensitivity and specificity were 75.5 and 88.2%, respectively. Patients who had Hb values of 12 or below who developed HFS had longer median times without HFS compared to patients with high Hb values (115 vs. 75 days, p = 0.30, hazard ratio = 1.42, 95% CI 0.73-2.76) and a greater area under the Kaplan-Meier curves (p < 0.05). CONCLUSION: This research suggests that the Hb value is an important factor for developing HFS.

Retrospective Analysis of the Risk Factors for Grade IV Neutropenia in Oesophageal Cancer Patients Treated with a Docetaxel, Cisplatin, and 5-Fluorouracil Regimen.

BACKGROUND: Previous Japanese trials of the docetaxel, cisplatin, and 5-fluorouracil regimen for oesophageal cancer have demonstrated that a large proportion of patients also develop grade IV neutropenia. Our aim was to examine the risk factors for neutropenia in patients treated with this regimen. METHODS: We retrospectively analysed the risk factors for developing grade IV neutropenia in 66 patients with oesophageal cancer using a multivariate analysis. RESULTS: After administering the docetaxel, cisplatin, and 5-fluorouracil regimen, 49 patients (74.2%) developed grade IV neutropenia. Grade IV neutropenia was significantly associated with platelet count (p < 0.01), alanine transaminase level (p = 0.05), and proton-pump inhibitor administration (p < 0.05). Receiver operating characteristic curve analysis confirmed a platelet count of 290 × 103/µL as the optimal diagnostic cut-off value for grade IV neutropenia. The receiver operating characteristic area for grade IV neutropenia was increased by including patients that were administered a proton-pump inhibitor and alanine transaminase level (updated model; sensitivity and specificity, 75.5 and 88.2%, respectively). CONCLUSIONS: Our findings suggest that a platelet count is the most significant predictor of grade IV neutropenia.

Transient GPI-anchored protein homodimers are units for raft organization and function.

Advanced single-molecule fluorescent imaging was applied to study the dynamic organization of raft-associated glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the plasma membrane and their stimulation-induced changes. In resting cells, virtually all of the GPI-APs are mobile and continually form transient (~200 ms) homodimers (termed homodimer rafts) through ectodomain protein interactions, stabilized by the presence of the GPI-anchoring chain and cholesterol. Heterodimers do not form, suggesting a fundamental role for the specific ectodomain protein interaction. Under higher physiological expression conditions , homodimers coalesce to form hetero- and homo-GPI-AP oligomer rafts through raft-based lipid interactions. When CD59 was ligated, it formed stable oligomer rafts containing up to four CD59 molecules, which triggered intracellular Ca(2+) responses that were dependent on GPI anchorage and cholesterol, suggesting a key part played by transient homodimer rafts. Transient homodimer rafts are most likely one of the basic units for the organization and function of raft domains containing GPI-APs.

Efficient selection of knocked-in pluripotent stem cells using a dual cassette cellular elimination system.

Although recent advances in genome editing technology with homology-directed repair have enabled the insertion of various reporter genes into the genome of mammalian cells, the efficiency is still low due to the random insertion of donor vectors into the host genome. To efficiently select knocked-in cells without random insertion, we developed the "double-tk donor vector system," in which the expression units of the thymidine kinase of herpes simplex virus (HSV-tk) are placed on both outer sides of homology arms. This system is superior in enriching knocked-in human induced pluripotent stem cells (hiPSCs) than conventional donor vector systems with a single or no HSV-tk cassette. Using this system, we efficiently generated fluorescent reporter knockin hiPSCs targeting POU5F1 (OCT3/4), EEF1A1, H2BC21 (H2B clustered histone 21), ISL1, and MYH7 genes. These results indicate that the double-tk donor vector system enables efficient selection of knocked-in hiPSCs carrying reporter proteins.

Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice.

Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF-ß, and Ang-1,2. Among these, the role of VEGF-A signaling in vessel morphogenesis is best understood. The biological activity of VEGF-A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF-A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1-tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1-tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development.

Environmental polarity estimation in living cells by use of quinoxaline-based full-colored solvatochromic fluorophore PQX and its derivatives.

Fluorosolvatochromic probes have recently attracted interest for live cell imaging. We recently tested our quinoxaline-based fluorosolvatochromic probes, PQX and MPQX, for in vivo imaging applications. PQX and MPQX are characterized by donor (pyrrole site)-acceptor (quinoxaline site) structure, and the peak emission wavelength of these compounds varies over the visible wavelength range depending on the polarity of the solvent used, for a variety of solvents from hexane to water. A linear relationship was obtained between peak emission wavenumber and E(T)(N) (normalized solvent polarity). When tested on cultured HEp-2 cells, the results showed that the PQX and MPQX were not harmful at the applied concentrations (10(-5) M), and site-dependent fluorescence spectra in living cells were observed with PQX treatment, which indicates that PQX penetrates into the plasma membrane, followed by delocalization throughout the cells. MPQX was also able to penetrate the cell membrane, distribute throughout the cells and emit fluorescence, but did not show site-dependent intracellular fluorescence spectra. These results suggest that PQX is a remarkable tool for investigating the local polarity environment in cells after appropriate conjugation to biomolecules.

Correlation between the consumption of meropenem or doripenem and meropenem susceptibility of Pseudomonas aeruginosa in a university hospital in Japan.

The appropriate use of carbapenems is essential in order to prevent resistance in Pseudomonas aeruginosa. We investigated the correlation between the consumption of meropenem or doripenem and the susceptibility of P. aeruginosa to meropenem in a Japanese university hospital from 2004 to 2009. The susceptibility data of P. aeruginosa and the annual consumption of meropenem or doripenem were analyzed. The consumption of meropenem or doripenem was calculated using the Anatomic Therapeutic Chemical classification and defined daily doses methodology. Meropenem consumption decreased and doripenem consumption increased and throughout the entire investigation period, total consumption of meropenem plus doripenem was stable. Although the annual number of isolated P. aeruginosa has not changed, the annual number of isolated multidrug resistant P. aeruginosa decreased by measures against nosocomial infection. The rate of meropenem resistant P. aeruginosa decreased in a time-dependent manner. Meropenem consumption was positively correlated with the meropenem resistance rate among P. aeruginosa (r = 0.9455, p<0.01). The total consumption of meropenem and doripenem was not correlated with the meropenem resistance rate (r = -0.6601, p>0.1). These results suggested that even if the total consumption of meropenem plus doripenem was not changed, meropenem susceptibility among P. aeruginosa improved by the decrease of meropenem consumption. Although meropenem and doripenem have been suggested to show cross-resistance with each other, the reduction of meropenem consumption might be effective for preventing an increase of meropenem-resistant P. aeruginosa.

Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice.

Kaede is a photoconvertible fluorescence protein that changes from green to red upon exposure to violet light. The photoconversion of intracellular Kaede has no effect on cellular function. Using transgenic mice expressing the Kaede protein, we demonstrated that movement of cells with the photoconverted Kaede protein could be monitored from lymphoid organs to other tissues as well as from skin to the draining lymph node. Analysis of the kinetics of cellular movement revealed that each subset of cells in the lymph node, such as CD4(+) T, CD8(+) T, B, and dendritic cells, has a distinct migration pattern in vivo. Thus, the Kaede transgenic mouse system would be an ideal tool to monitor precise cellular movement in vivo at different stages of immune response to pathogens as well as in autoimmune diseases.

Generation of two ISL1-tdTomato reporter human induced pluripotent stem cell lines using CRISPR-Cas9 genome editing.

ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.

Albino mice with the point mutation at the tyrosinase locus show high cholesterol diet-induced NASH susceptibility.

Non-alcoholic fatty liver disease (NAFLD) constitutes a metabolic disorder with high worldwide prevalence and increasing incidence. The inflammatory progressive state, non-alcoholic steatohepatitis (NASH), leads to liver fibrosis and carcinogenesis. Here, we evaluated whether tyrosinase mutation underlies NASH pathophysiology. Tyrosinase point-mutated B6 (Cg)-Tyrc-2J/J mice (B6 albino) and C57BL/6J black mice (B6 black) were fed with high cholesterol diet (HCD) for 10 weeks. Normal diet-fed mice served as controls. HCD-fed B6 albino exhibited high NASH susceptibility compared to B6 black, a phenotype not previously reported. Liver injury occurred in approximately 50% of B6 albino from one post HCD feeding, with elevated serum alanine aminotransferase and aspartate aminotransferase levels. NASH was induced following 2 weeks in severe-phenotypic B6 albino (sB6), but B6 black exhibited no symptoms, even after 10 weeks. HCD-fed sB6 albino showed significantly higher mortality rate. Histological analysis of the liver revealed significant inflammatory cell and lipid infiltration and severe fibrosis. Serum lipoprotein analysis revealed significantly higher chylomicron and very low-density lipoprotein levels in sB6 albino. Moreover, significantly higher small intestinal lipid absorption and lower fecal lipid excretion occurred together with elevated intestinal NPC1L1 expression. As the tyrosinase point mutation represents the only genetic difference between B6 albino and B6 black, our work will facilitate the identification of susceptible genetic factors for NASH development and expand the understanding of NASH pathophysiology.

Utilization of a novel Sendai virus vector in ex vivo gene therapy for hemophilia A.

Sendai virus (SeV) vectors are being recognized as a superior tool for gene transfer. Here, we report the transfection efficacy of a novel, high-performance, replication-defective, and persistent Sendai virus (SeVdp) vector in cultured cells and in mice using a near-infrared fluorescent protein (iRFP)-mediated in vivo imaging system. The novel SeVdp vector established persistent infection, and strong expression of inserted genes was sustained indefinitely in vitro. Analysis of iRFP-expressing cells transplanted subcutaneously into NOG, nude, and ICR mice suggests that innate immunity was involved in the exclusion of the transplanted cells. We also evaluated the feasibility of this novel SeVdp vector for hemophilia A gene therapy. This system enabled insertion of full-length FVIII genes, and transduced cells secreted FVIII into the culture medium. Transient FVIII activity was detected in the plasma of mice after intraperitoneal transplantation of these FVIII-secreting cells. Further improvement in methods to evade immunity, such as simultaneous expression of immunomodulatory genes, would make this novel vector a very useful tool in regenerative medicine.

Impact of antimicrobial stewardship by infection control team in a Japanese teaching hospital.

The objective of this study is to investigate an effect on the antimicrobial appropriate use of the antimicrobial stewardship. We investigated the consumption of injectable antimicrobials from 2001 to 2007 and the administration period of specific antimicrobials (carbapenems, fourth-generation cephalosporins, anti-methicillin-resistant Staphylococcus aureus (MRSA) agents) in the individual patient. Since September 2004, the infection control team at Osaka University Hospital has been promoting appropriate use of antimicrobials through consultation, education, and weekly surveillance of specific antimicrobial usage. We obtained the amount of all antimicrobial injections as titer from the medical information database of the electronic medical chart system retrospectively. Antimicrobial use densities (AUD) were evaluated by measuring the number of doses administered/1000 patient-days. Although the number of inpatient admissions and operations increased 1.53- and 1.39-fold, respectively, in the seven years from 2001 to 2007, the expenditure on specific antimicrobials decreased markedly with AUD of specific antimicrobials decreasing from 39.6 to 29.2. The percentage of inpatients receiving specific antimicrobials decreased from 19.8% to 9.8%, and the ratio of the number of inpatients administered a specific antimicrobial within seven days to the number of inpatients administered each specific antimicrobial increased to over 60%. This led to reduction in the total expenditure of antimicrobials by about 100 million yen annually. The incidence of hospital-associated MRSA (HA-MRSA) infection decreased from 0.93% (2003) to 0.68% (2007). We can reduce the expenditure of antimicrobials without increasing incidence of the HA-MRSA by antimicrobial stewardship, and we think that appropriate use of antimicrobials is achieved progressively.

Evaluation of environmental contaminations and occupational exposures involved in preparation of chemotherapeutic drugs.

Many healthcare workers are concerned about the risk of occupational exposures to hazardous drugs. The Japanese Society of Hospital Pharmacists (JSHP) revised the "Guidelines for the Handling of Antineoplastic Drugs in Hospitals", however, the precautions and awareness of handling drugs varied in institutions. We assessed the levels of environmental contaminations in our hospital and urinary excretion of cyclophosphamide (CP) and ifosfamide (IF) in pharmacists and nurses. In environmental studies, we obtained samples by wiping the surfaces around two biological safety cabinets (BSCs) on eight days for four months. One BSC was equipped in hospital pharmacy and the other was equipped in an oncology ward, and used for preparing chemotherapeutic drugs for outpatients and for inpatients, respectively. We obtained the urine samples from 6 pharmacists and 2 nurses. We used solid phase extraction (SPE) as a convenient extraction procedure and liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) for the analysis of the samples. CP was detected on the working surfaces inside both BSCs, and detected at low levels on the back surfaces of the BSCs and at the working tables around the BSCs. IF over the LLOQ was not detected in both BSCs. CP and IF were not detected in all urine samples of pharmacists and nurses. Detection frequencies and amounts of these drugs were low levels, compared with previous reports in Japan, and our results showed that improving awareness about handling hazardous drugs could reduce the risk of the occupational exposures.

Epstein-Barr virus-derived vector suitable for long-term expression in neurons.

Exogenous gene expression is a fundamental and indispensable technique for testing gene function in neurons. Several ways to express exogenous genes in neurons are available, but each method has pros and cons. The lentivirus vector is useful for high efficiency gene transfer to neurons and stabilizes gene expression via genome integration, but this integration may destroy the host genome. The Epstein-Barr virus (EBV)-derived vector (EB vector) is an accessible and useful vector in human cell lines because the vector is not integrated into the host genome but stays in the nucleus as an episome. However, there has been no report on this process in rodent neurons. We examined the usefulness of the EB vector for testing gene function in neurons. We found that EB vector-derived exogenous proteins such as green fluorescent protein (GFP) and GFP-tagged actin were easily detectable even after three weeks of transfection. Second, a tetracycline-induced gene expression system in the EB vector was active after three weeks of transfection, indicating that plasmids were retained in neurons for up to three weeks. Third, we determined that only Family of repeat element of the plasmid vector is essential for its long-term presence in neurons. These results show that the modified EB vector is a useful tool for examining gene function in neurons.

Tumor-suppressive lipoxygenases inhibit the expression of c-myc mRNA coding region determinant-binding protein/insulin-like growth factor II mRNA-binding protein 1 in human prostate carcinoma PC-3 cells.

8S-Lipoxygenase (8S-LOX) is known as a mouse homolog of human 15S-LOX-2. 15S-LOX-2 was down-regulated in malignant transformation of prostate epithelial cells, and its overexpression caused cell cycle arrest. To determine whether 8S-LOX would have a growth inhibitory effect on prostate carcinoma, we obtained human prostate carcinoma PC-3 cells expressing 8S-LOX or 15S-LOX-2. The growth rate of cells measured by colorimetric assay was reduced by expression of 8S-LOX and 15S-LOX-2. The addition to enzyme-expressing cells of arachidonic acid enhanced the growth suppressive effect, whereas the expression of catalytically inactive mutants did not affect cell growth, suggesting that the effect was product-dependent. DNA microarray and quantitative reverse transcription-PCR analyses revealed that the c-myc mRNA coding region determinant-binding protein/insulin-like growth factor II mRNA-binding protein 1 (CRD-BP/IMP-1), known as an oncofetal protein, was down-regulated in 8S-LOX- and 15S-LOX-2-expressing PC-3 cells. Targeted knockdown of CRD-BP/IMP-1 resulted in inhibition of the DNA synthesis rate of PC-3 cells as measured by bromodeoxyuridine incorporation. We propose that expression of 8S-LOX and 15S-LOX-2 suppresses CRD-BP/IMP-1 expression, resulting in inhibition of human prostate carcinoma PC-3 cell proliferation.