Grant amount：\4290000 （ Direct Cost: \3300000 、 Indirect Cost：\990000 ）

MRSA infection is an ongoing challenge. Staphylococcal species exchange genetic information through staphylococcal cassette chromosome (SCC). It encompasses not only drug resistance genes but also diverse genes useful for better adaptation to the unfriendly environment. The present study has aimed to reveal how the mobile genetic elements emerged, evolved, and spread among staphylococcal species. SCC elements obtained from MRSA clinical isolates were determined for their nucleotide sequences, and compared with staphylococcal genomes deposited in a public databank. Following two novel elements were identified: a novel subtype of type IX SCC carrying mec gene (SCCmec) in Myanmar MRSA, an SCC in MRSA from including speG and a staphylococcal surface protein coding gene (sasG) in Japanese MRSA. Obtained data suggests that they have originated in coagulase-negative staphylococcal species.

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Grant amount：\17940000 （ Direct Cost: \13800000 、 Indirect Cost：\4140000 ）

In three countries in Asia and Caribbean regions (Bangladesh, Myanmar, and Cuba) where information on antimicrobial resistance (AMR) is insufficient, resistance rates to various antimicrobials, prevalence of genes associated with AMR and virulence, genetic characteristics of drug-resistant isolates were analyzed for major pathogenic bacterial species (Escherichia coli, Klebsiella pneumoniae, Acinetobacter, Staphylococccus aureus, Enterococcus). As a result, it was revealed that various beta-lactamase genes have been widely distributed to Gram-negative rods represented by E. coli, and virulence factor PVL (Panton-Valentine leukocidin) associated with increased pathogenicity has been prevalent at relatively high rate among methicillin-resistant S. aureus (MRSA).

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Grant amount：\4810000 （ Direct Cost: \3700000 、 Indirect Cost：\1110000 ）

After the introduction of the pneumococcal conjugate vaccine (PCV) for children, an increase in pneumococcal diseases caused by non-PCV serotypes is a worldwide concern. The prevalence of serotypes, antimicrobial resistance, and its relevance to PspA which is a considered as promising vaccine candidate were investigated for 729 (678 children, 51 adults) non-invasive pneumococcal isolates. Isolates were collected in hospitals and clinics in Hokkaido between June and November 2016. As a results, 87.9% of isolates from children belonged to non-PCV13 serotypes and most isolates (81-100%) of the dominant serotypes 15A, 35B and 23A were non-susceptible to penicillin. While most of the isolates (99%) had PspA belonging to either family 1 or 2, PspA family 3/clade 6 was detected in only non-PCV13 serotype 37 isolates assigned to ST447 and ST7970 showing the mucoid phenotype.

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Grant amount：\4940000 （ Direct Cost: \3800000 、 Indirect Cost：\1140000 ）

The staphylococcal cassette chromosome (SCC) is a bacterial mobile genetic element inserted in the genome of staphylococcal species. SCC contains a variety of functional genes that provide biological benefits to the host bacteria. Staphylococcal species exchange genetic information by horizontal gene transfer of SCCs, which enables rapid adaption to changing environments. Three ST5-MRSA strains (SC640, SC792, and SC955) positive for both ACME-arc and speG were subjected to whole-genome sequencing, and their SCC composite islands (SCC-CIs) were determined. The SCC-CIs of these strains contained following novel SCCs: novel SCCs containing speG gene (SC640, SC792, and SC955), a novel SCCmec type XIV (SC792), and a truncated SCCmec type XIV-related structure (SC640).

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Grant amount：\4940000 （ Direct Cost: \3800000 、 Indirect Cost：\1140000 ）

Methicillin-resistant Staphylococcus aureus (MRSA) is drug-resistant bacteria known for a long time. Recently, two MRSA clones with novel genetic traits (①MRSA harboring SCCmec-IVl which is a novel genetic complex; ②Coagulase type-II MRSA having ACME which is a genetic complex related to attachment to human skin) have been reported. We investigated their prevalence in clinical isolates in Hokkaido, Japan. These MRSA ①, ② were detected in 3.5% and 5.1% of isolates from outpatients of community, and 3% and 0.9% of isolates from a university hospital, respectively. Genetic diversity was found in MRSA with ACME, and their spread in the community and hospitals was concerned.

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Grant amount：\17550000 （ Direct Cost: \13500000 、 Indirect Cost：\4050000 ）

Epidemic dynamics for long periods of human rotaviruses in Asia was analyzed molecular epidemiologically based on whole genome (11 gene segments). In Wuhan, China, G3P[8] was the most prevalent genotype of rotaviruses over past 10 years, which was thereafter replaced by G9P[8]. Change of lineages was detected for most of their gene segments with the passage of time. In Bangladesh, two lineages of the most prevalent G2P[4] genotype in 2010 were replaced by a novel lineage after 3 years. These findings indicated that replacement of gene segments via reassortment and mutation occurred over time among rotaviruses with prevalent genotypes. It was suggested that genetic changes have been occurring constantly even among the same genotype of rotaviruses.

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Grant amount：\5460000 （ Direct Cost: \4200000 、 Indirect Cost：\1260000 ）

The Arginine catabolic mobile element (ACME), a genomic island identified in the Staphylococci genome, is characteristically found in strain USA300, which is the predominant MRSA clone in the USA and is spreading globally. In the present study, the prevalence of ACME and the genetic diversity of ACME (+) MRSA were investigated for clinical isolates collected in Hokkaido.
The percentage of ACME carriage against the all MRSA isolates significantly increased in the course of study. Increased number of MRSA with the identical genotype as USA300 was observed, which suggests its potential spread in northern Japan. We focused on the ACME (+) MRSA with type II SCCmec (MRSA-II) because MRSA-II is the predominant genotype in Japanese MRSA. The genetic structures of ACME and its flanking region in MRSA-II were determined and novel genetic variants were identified. The similar analysis was carried out on coagulase-negative staphylococci clinical isolates obtained in Hokkaido.

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Grant amount：\5460000 （ Direct Cost: \4200000 、 Indirect Cost：\1260000 ）

To investigate prevalence in Japan of community-acquired MRSA (CA-MRSA) which is concerned for its global spread, and their molecular epidemiological characteristics, we analyzed clinical isolates from the Sapporo Medical University Hospital (601 isolates, 2008-2010) and those from local hospitals/clinics in Hokkaido prefecture (outpatients, 422 isolates in 2011). As a result, SCCmec types IV and V, which are genetic markers of CA-MRSA, were detected in 4.3% and 17.1% in the isolates from the University Hospital and local hospitals/clinics, respectively. Among them, seven isolates were revealed to be USA300 clone which is dominant in the US (ST8, positive for PVL and ACME), and many ST5 MRSA having ACME were detected. These emerging CA-MRSA clones were confirmed to be distributed in Japan.

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Grant amount：\19500000 （ Direct Cost: \15000000 、 Indirect Cost：\4500000 ）

To elucidate genetic characteristics of new variants and emerging types of rotavirus that spread to mainly in Asian countries, whole genomic sequence analysis and phylogenetic analysis were conducted. In China and Bangladesh, the predominant genotypes were G1P[8]/G3P[8], G2P[4], respectively, among which reassortant viruses containing gene segments derived from animal rotavirus were detected. Rotaviruses with P[8]b, a new variant of P[8]-VP4 genotype, were detected in China, Bangladesh, and Myanmar, and mostly associated with globally emerging type G9. Whole genomic analysis of various rotaviruses strains with emerging genotypes from Asia and other regions (genotypes G1P[6], G2P[6], G3P[2]、G3P[6], G3P[9], G4P[10], G6P[9], G8P[1], G9P[19]) revealed that these strains could be classified into 1)reassortant between different human rotavirus genogroups, 2)reassortant between human and animal rotaviruses, and 3) animal rotaviruses that directly transmitted to humans. These findings indicated that reassortment and interspecies transmission may be the main causing mechanisms of new variants and emergingtypes of human rotaviruses.

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Grant amount：\4810000 （ Direct Cost: \3700000 、 Indirect Cost：\1110000 ）

Prevalence and molecular epidemiologic characteristics of methicillin-resistant S.aureus (MRSA) were analyzed for numerous Staphylococcus aureus isolates derived from outpatients in hospitals in Hokkaido. As a result, several MRSA strains (genotypes ST6, ST59) which harbor the Panton-Valentine Leukocidin (PVL) gene typical to CA-MRSA, and the genetic element ACME unique to USA300 CA-MRSA clone predominating in USA,were identified, suggesting the wide distribution of these MRSA to the community.

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Grant amount：\4550000 （ Direct Cost: \3500000 、 Indirect Cost：\1050000 ）

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Grant amount：\3300000 （ Direct Cost: \3300000 ）

成果の一部を上げる. まず, ニジマス由来RTG-2細胞の環境ストレスに対する応答を解析した. 化学汚染物質トリブチルスズ(TBT)がRTG-2にMAP kinase (ERK, JNK, p38 MAP kinase)の活性化を惹起すること, caspase活性化を伴うapoptosisを誘導することを明らかにした. さらにJNKがTBTによるcaspase依存的なapoptosisに, p38 MAP kinaseがcaspase非依存的な細胞死に関与することを明らかにした. 更に熱ショックに対する応答の解析を行った.
次に, 生物進化の過程でヒトを含めて広く保存されてきた様々な環境ストレスに対して共通する検知・応答機構について, 細胞骨格と細胞接着に注目して検討を進めた. 即ち, 良く保存されているArf-GAPタンパクGIT1とアダプタータンパクNckの結合様態と生理的意義について, 分子細胞生物学的な検討を行い, GIT1のチロシンリン酸化がNickを結合パートナーとして細胞内局在の移動をもたらし細胞機能を発揮していることが示唆された. これらのタンパクは線虫から哺乳動物まで重要な役割を果たしており, 普遍性の高いものであることが推察される.

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Grant amount：\3500000 （ Direct Cost: \3500000 ）

Because we have performed numerous experiments, we would like to focus on two following projects..In 2005, using specific antibodies against isoforms of WAVE, we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. We also found that all 3 WAVE isoforms are substrates for calpain in vivo and in vitro. Although portions of these 3 isoforms were commonly distributed in the actin-and Arp2/3-rich edge of the lamellipodia in spreading platelets, only WAVE2 remained in the cell fringe following detergent extraction or fixation of the cells. These data suggest that the 3 WAVE isoforms exhibit common and distinct features and may potentially be involved in the regulation of actin cytoskeleton in platelets. In 2006, we also found that platelets express IRSp53 protein. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP(3)-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP(3).

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