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[Introduction] Bloodstream infection (BSI) is a serious complication of HCT that may be life-threatening. BSI frequently occurs before neutrophil engraftment (pre-engraftment BSI), but has also been reported after neutrophil recovery (post-engraftment BSI). In contrast to pre-engraftment BSI, the clinical features and risk factors of post-engraftment BSI remain unclear. [Aims] We investigated the clinical characteristics of and risk factors for post-engraftment BSI. [Methods] This retrospective study included 176 adult patients who underwent HCT and achieved neutrophil engraftment between 2006 and 2017 at our institute. Diagnoses consisted of AML (n=86), ALL (n=36), MDS (n=21), MPN (n=4), CML (n=1), ATL (n=6), aplastic anemia (n=6), and malignant lymphoma (n= 16). The median age at HCT was 42 y (range 16-67 y). Graft sources were BM (n=69), PB (n=57), and CB (n=50). Fifty-five patients (31.2%) had a high (≥3) HCT-CI score on HCT. Sixty-four patients (36.4%) received HCT with an advanced disease status. Fluoroquinolone (FQ) as prophylaxis was administered to 89 patients (50.6%). Central venous catheters (CV) were inserted in all patients before HCT. All patients consulted a dentist before HCT and received guidance on appropriate oral self-care to prevent severe oral mucositis; 92 (52.3%) continuously received intensive oral care after HCT (I-care; visit to a dentist at least once a week until neutrophil engraftment for the assessment of oral mucositis and cleaning), whereas 84 (47.7%) only performed oral self-care (S-care; according to guidance by a dentist). In the present study, BSI was defined as an infectious state with fever (≥38°C) and the isolation of a pathogen on at least 1 blood culture or on 2 or more if a common skin contaminant was isolated. Post-engraftment BSI was evaluated until day 180. [Results] Seventy-five events of BSI (in 69 patients) occurred until day 180. The total cumulative incidence of BSI (CIB) was 39.2%. The CIB of pre- and post-engraftment BSI were similar at 21.6% (n=38) and 21.0% (n=37) (p=1.0), respectively. Six patients developed pre- and post-engraftment BSI. CV was inserted in all patients when BSI occurred. Twenty-five pathogens were isolated in the present study. Regarding the type of pathogen, Gram-positive cocci were the most common in pre-/post-engraftment BSI (63.2%/69.0%). Gram-negative (29.0%/14.3%) and -positive rods (15.8%/19.0%) were detected. Staphylococcus epidermidis was the most frequently detected species in pre/post-engraftment BSI (31.6%/57.1%). Similar to CIB, no significant difference was observed in the pathogens identified between pre-/post-engraftment BSI (p=0.34). We performed further analyses to identify risk factors for post-engraftment BSI. In Fisher's exact test as a univariate analysis, HCT-CI≥3 (p=0.045), TBI≥3 Gy (p=0.041), not administered FQ (p=0.042), no I-care (p=0.003), and a graft source of BM (p=0.002) were identified as risk factors for post-engraftment BSI. Age (p=0.25), conditioning (p=0.197), repeated HCT (0.607), disease stage prior to HCT (p=0.701), and a history of pre-engraftment BSI (p=0.501) did not contribute to post-engraftment BSI. A logistic regression test with backward stepwise selection based on p-values as the multivariate analysis revealed that I-care (OR 0.358, 95%CI: 0.16-0.801, p=0.0124) and a graft source of PB (OR 0.322, 95%CI: 0.124-0.837, p=0.02) reduced the risk of post-engraftment BSI. Furthermore, to confirm the impact of the oral care type on post-engraftment BSI, we compared the CIB of the S- and I-care groups. The CIB of pre-engraftment BSI was similar between the I- and S-care groups (21.4% vs 21.7%, p=1), whereas that of post-engraftment BSI was significantly lower in the I-care group (12.0% vs 29.8%, p [Conclusion] CIB and isolated pathogens were similar between pre- and post-engraftment BSI, even if neutrophils had recovered sufficiently. Since BSI may occur at any time during transplantation, careful follow-ups are needed. Intensive oral care by a dental specialist may reduce the risk of post-engraftment BSI. Disclosures No relevant conflicts of interest to declare.

The crystal structure of an uncharacterized protein TTHA0061 from Thermus thermophilus HB8, was determined and refined to 1.8 A by a single wavelength anomalous dispersion (SAD) method. The structural analysis and comparison of TTHA0061 with other existing structures in the Protein Data Bank (PDB) revealed a novel fold, suggesting that this protein may belong to a translation initiation factor or ribosomal protein family. Differential scanning calorimetry analysis suggested that the thermostability of TTHA0061 increased at pH ranges of 5.8–6.2, perhaps due to the abundance of glutamic acid residues.

This paper argues for the rise of a functional category Pred(ication) in the sense of Bowers (1993) in the history of English by investigating the distribution of as, floating quantifiers, expletive it, and inversion of subjects and predicates in small clauses. The results of this investigation reveal that evidence for the presence of Pred in small clauses began to be attested in the fourteenth century. This indicates that they have undergone a change from the structure headed by a lexical category to the structure headed by Pred, which is shown to have been completed during the eighteenth century.

Summary Sister chromatid separation is initiated at anaphase onset by the activation of separase, which removes cohesins from chromosomes. However, it remains elusive how sister chromatid separation is completed along the entire chromosome length. Here we found that, during early anaphase in Saccharomyces cerevisiae, sister chromatids separate gradually from centromeres to telomeres, accompanied by regional chromosome stretching and subsequent recoiling. The stretching results from residual cohesion between sister chromatids, which prevents their immediate separation. This residual cohesion is at least partly dependent on cohesins that have escaped removal by separase at anaphase onset. Meanwhile, recoiling of a stretched chromosome region requires condensins and generates forces to remove residual cohesion. We provide evidence that condensins promote chromosome recoiling directly in vivo, which is distinct from their known function in resolving sister chromatids. Our work identifies residual sister chromatid cohesion during early anaphase and reveals condensins' roles in chromosome recoiling, which eliminates residual cohesion to complete sister chromatid separation., Graphical Abstract Highlights ► Sister chromatid cohesion partially persists after anaphase onset in budding yeast ► Residual cohesion is partly dependent on cohesins and causes chromosome stretching ► Condensins directly promote recoiling of stretched chromosomes during early anaphase ► Chromosome recoiling generates forces to remove residual sister chromatid cohesion

Since the realisation that the antigen-binding regions of antibodies, the variable (V) regions, can be uncoupled from the rest of the molecule to create fragments that recognise and abrogate particular protein functions in cells, the use of antibody fragments inside cells has become an important tool in bioscience. Diverse libraries of antibody fragments plus in vivo screening can be used to isolate single chain variable fragments comprising VH and VL segments or single V-region domains. Some of these are interfering antibody fragments that compete with protein-protein interactions, providing lead molecules for drug interactions that until now have been considered difficult or undruggable. It may be possible to deliver or express antibody fragments in target cells as macrodrugs per se. In future incarnations of intracellular antibodies, however, the structural information of the interaction interface of target and antibody fragment should facilitate development of binding site mimics as small drug-like molecules. This is a new dawn for intracellular antibody fragments both as macrodrugs and as precursors of drugs to treat human diseases and should finally lead to the removal of the epithet of the 'undruggable' protein-protein interactions.

Kinetochore capture and transport by spindle microtubules plays a crucial role in high-fidelity chromosome segregation, although its detailed mechanism has remained elusive. It has been difficult to observe individual kinetochore–microtubule interactions because multiple kinetochores are captured by microtubules during a short period within a small space. We have developed a method to visualize individual kinetochore–microtubule interactions in Saccharomyces cerevisiae, by isolating one of the kinetochores from others through regulation of the activity of a centromere. We detail this technique, which we call ‘centromere reactivation system’, for dissection of the process of kinetochore capture and transport on mitotic spindle. Kinetochores are initially captured by the side of microtubules extending from a spindle pole, and subsequently transported poleward along them, which is an evolutionarily conserved process from yeast to vertebrate cells. Our system, in combination with amenable yeast genetics, has proved useful to elucidate the molecular mechanisms of kinetochore–microtubule interactions. We discuss practical considerations for applying our system to live cell imaging using fluorescence microscopy.

Sapovirus (SaV) is an important pathogen of human acute gastroenteritis. A gastroenteritis outbreak occurred at a wedding hall in October 2007 in Ehime Prefecture, Japan. One hundred nine people who had either attended wedding parties or had eaten a box lunch at a conference held at the same hall complained of gastroenteritis symptoms. Among these 109 people, stool specimens from 56 patients were available for pathogen screening, and 20 (35.7%) of these specimens were positive for SaV, of whom 18 showed symptoms. The numbers of cDNA copies of the specimens ranged from 2.36 x 10(6) to 3.03 x 10(10) for symptomatic patients, and 2.19 x 10(6) and 1.18 x 10(9) per gram of stool for two asymptomatic food handlers. The incubation periods of the 18 symptomatic patients ranged from 14.5 to 99.5 hr. Identical nucleotide sequence types of SaV; that is, a single synonymous nucleotide difference (transition) or microheterogeneity, was detected in stool specimens from the symptomatic patients and the asymptomatic food handlers, with the direct nucleotide sequence of approximately 2.3 kb 3' end of the genome. Based on the phylogenetic analysis with the complete capsid nucleotide sequence, these strains were clustered into genogroup IV. This outbreak was thought to be caused by a single source, and underscores the importance of proper hygiene in the environment and/or in food-handling practices to control SaV outbreaks.

Summary In early mitosis, microtubules can be generated at kinetochores as well as at spindle poles. However, the role and regulation of kinetochore-derived microtubules have been unclear. In general, metaphase spindle microtubules are oriented such that their plus ends bind to kinetochores. However, we now have evidence that, during early mitosis in budding yeast, microtubules are generated at kinetochores with distal plus ends. These kinetochore-derived microtubules interact along their length with microtubules that extend from a spindle pole, facilitating kinetochore loading onto the lateral surface of spindle pole microtubules. Once kinetochores are loaded, microtubules are no longer generated at kinetochores, and those that remain disappear rapidly and do not contribute to the metaphase spindle. Stu2 (the ortholog of vertebrate XMAP215/ch-TOG) localizes to kinetochores and plays a central role in regulating kinetochore-derived microtubules. Our work provides insight into microtubule generation at kinetochores and the mechanisms that facilitate initial kinetochore interaction with spindle pole microtubules., Graphical Abstract Highlights ► A short-lived population of kinetochore-derived microtubules (MTs) has distal plus ends ► These MTs are generated in early mitosis and regulated by Stu2 ► They interact with spindle pole-derived MTs before kinetochore attachment ► Once kinetochores attach to spindle-pole MTs, the short-lived MT population disappears

The maturation of [NiFe]-hydrogenases is a catalyzed process involving the activities of at least seven proteins. The last step consists of the endoproteolytic cleavage of the precursor of the large subunit, after the [NiFe]-metal center has been assembled. The HycI endopeptidase is involved in the C-terminal processing of HycE, the large subunit of hydrogenase 3 from Escherichia coli . Although HycI has been well characterized biochemically, the crystallization of the protein has been quite challenging. Here, we present the crystal structure of HycI at 1.70 A resolution. The crystal structure resembles the recently reported solution structure (NMR) of the same protein and the holo-HyPD structure of the same family, but a significant conformational change is observed at the L5 loop, as compared with the solution structures of HycI and HyPD. In our crystal structure, three specific metal binding sites (Ca1–3) were identified and these metal ions are possibly involved in the C-terminal cleavage of HycE.

Duplication of chromosomal DNA is a temporally and spatially regulated process. The timing of DNA replication initiation at various origins is highly coordinated; some origins fire early and others late during S phase. Moreover, inside the nuclei, the bulk of DNA replication is physically organized in replication factories, consisting of DNA polymerases and other replication proteins. In this review article, we discuss how DNA replication is organized and regulated spatially within the nucleus and how this spatial organization is linked to temporal regulation. We focus on DNA replication in budding yeast and fission yeast and, where applicable, compare yeast DNA replication with that in bacteria and metazoans.

Three novel C 27 N 3 -type Lycopodium alkaloids, lycochinines A–C ( 1 – 3 ) consisting of an octahydroquinoline or a decahydroquinoline, a quinolizidine, and a piperidine, were isolated from the club moss Lycopodium chinense . The relative stereochemistry of 1 – 3 was determined by a combination of NOESY correlations and chemical transformations.

ST1710, a member of the multiple antibiotic resistance regulator (MarR) family of regulatory proteins in bacteria and archaea, plays important roles in development of antibiotic resistance, a global health problem. Here, we present the crystal structure of ST1710 from Sulfolobus tokodaii strain 7 complexed with salicylate, a well-known inhibitor of MarR proteins and the ST1710 complex with its promoter DNA, refined to 1.8 and 2.10 A resolutions, respectively. The ST1710-DNA complex shares the topology of apo-ST1710 and MarR proteins, with each subunit containing a winged helix-turn-helix (wHtH) DNA binding motif. Significantly large conformational changes occurred upon DNA binding and in each of the dimeric monomers in the asymmetric unit of the ST1710-DNA complex. Conserved wHtH loop residues interacting with the bound DNA and mutagenic analysis indicated that R89, R90 and K91 were important for DNA recognition. Significantly, the bound DNA exhibited a new binding mechanism.

The evaporation process of multi-component fuel is different from one of a single component, because the properties of each component affects among the components. In actual engine, the spatial distribution of fuel vapor concentration dominates auto-ignition and initial combustion, and depends on the volatility and diffusivity of each component fuel contained in the multi-component fuel. Then, this study proposes a simplified numerical scheme for analysis of evaporation process of multi-component fuel sprays. Evaporation process is calculated by KIVA-II code based on the simple two-phases region that is approximated by modified saturated liquid-vapor line, which was obtained by connecting the 50% distillation temperature for each component under several pressure fields. Consequently, it can be quantitatively simulated that vapor of low boiling fuel component mostly exists around nozzle and spray tip region, the high boiling duel component, on the other hand, mostly appears near the spray tip. 기호설명p : 분사압력, MPaR : 반경방향 거리, mmT : 온도, KZ : 노즐로부터 거리, mm그리스문자 ρ : 주위기체 밀도, kg/m

Kinetochore components play a major role in regulating the transmission of genetic information during cell division. Ndc10p, a kinetochore component of the essential CBF3 complex in budding yeast is required for chromosome attachment to the mitotic spindle. ndc10-1 mutant was shown to display chromosome mis-segregation as well as an aberrant mitotic spindle ( Goh and Kilmartin, 1993 ). In addition, Ndc10p localizes along the spindle microtubules ( Muller-Reichert et al., 2003 ). To further understand the role of Ndc10p in the mitotic apparatus, we performed a three-dimensional electron microscopy (EM) reconstruction of mitotic spindles from serial sections of cryo-immobilized ndc10-1 mutant cells. This analysis reveals a dramatic reduction in the number of microtubules present in the half-spindle, which is connected to the newly formed spindle pole body (SPB) in ndc10-1 cells. Moreover, in contrast to wild-type (WT) cells, ndc10-1 cells showed a significantly lower signal intensity of the SPB components Spc42p and Spc110p fused with GFP, in mother cell bodies compared with buds. A subsequent EM analysis also showed clear defects in the newly formed SPB, which remains in the mother cell during anaphase. These results suggest that Ndc10p is required for maturation of the newly formed SPB. Intriguingly, mutations in other kinetochore components, ndc80-1 and spc24-1, showed kinetochore detachment from the spindle, similar to ndc10-1, but did not display defects in SPBs. This suggests that unattached kinetochores are not sufficient to cause SPB defects in ndc10-1 cells. We propose that Ndc10p, alongside its role in kinetochore–microtubule interaction, is also essential for SPB maturation and mitotic spindle integrity.

Genotype-specific cancer therapy promises to engender the era of personalised medicines in which rapid identification of tumour specific gene mutations coupled to rapid methods for efficacious drug identification will be applied. Aberrant signal transduction via protein-protein interactions is generally difficult to target with small molecules. However, macromolecules (macrodrugs) can be developed that interfere with protein-protein interactions by binding with high affinity and specificity to contact surfaces. Inhibitors of mutant RAS and its effector protein interactions affect cancer by attenuating aberrant RAS-dependent signal transduction and would be effective against mutant RAS in dividing cells of overt tumours and in putative cancer stem cells when they move into cell-cycle. Results with an antibody fragment blocking effector binding to RAS, illustrates that this is sufficient to prevent cancer. While macrodrugs have inherent problems of bio-distribution and delivery to target cells in patients, their efficacy suggests that efforts to achieve the goal of clinical use should be pursued.

To estimate the degree of crossbreeding between Japanese wild boars and crossbred Inobuta in Wakayama prefecture, we examined haplotypes of mitochondrial DNA (mtDNA) and genotypes of the nuclear glucosephosphate isomerase-processed pseudogene (GPIP) in tissue samples obtained from 176 wild boars over a 3-year period. Five different haplotypes (J10, J15, J21, J22 and J23) and 3 GPIP alleles (GPIP1, GPIP3a and GPIP3b) were detected. These genetic profiles were classified as Japanese wild boar lineage without the genetic markers typical of Inobuta. The present genetic profile of wild boars, consisting of mtDNA haplotypes and GPIP genotypes, is a useful tool for studying the genetic structure of the local feral population.

Although earlier pleuromutilin analogues showed potent in vitro antibacterial activity against some Gram-positive pathogens, their in vivo efficacy was low because of insufficient pharmacokinetic properties. We designed novel thioether pleuromutilin derivatives having a purine ring as a polar and water solubilizing group and identified a promising pleuromutilin analogue 6 with good solubility in water ( approximately 50 mg/mL). Compound 6 exhibited excellent in vitro and in vivo antibacterial activity against some Gram-positive strains, including drug-resistant pathogens.

Intracellular antibody fragments that interfere with molecular interactions inside cells are valuable in investigation of interactomes and in therapeutics, but their application demands that they function in the reducing cellular milieu. We show here a 2.7-A crystal structure of intracellular antibody folds based on scaffolds developed from intracellular antibody capture technology, and we reveal that there is no structural or functional difference with or without the intra-domain disulfide bond of the variable domain of heavy chain or the variable domain of light chain. The data indicate that, in the reducing in vivo environment, the absence of the intra-domain disulfide bond is not an impediment to correction of antibody folding or to interaction with antigen. Thus, the structural constraints for in-cell function are intrinsic to variable single-domain framework sequences, providing a generic scaffold for isolation of functional intracellular antibody single domains.

The emergence of bacterial resistance to multiple drugs poses a serious and growing health concern. Understanding and deciphering the mechanisms of these multiple drug resistance regulatory proteins through structural or biochemical means is an important endeavor. Here, we present the crystal structure of ST1710 from Sulfolobus tokodaii strain 7 in two different crystal forms, at 1.80 and 2.0A, respectively. The overall structure of the ST1710 dimer shares the topology of the MarR family of proteins, with each subunit containing a winged helix-turn-helix DNA-binding motif. We also show the protein-DNA interactions by biochemical methods. Our molecular modeling analysis suggested that Asp88 and Arg90 are the key residues in ST1710 involved in the protein-DNA interactions.

症例は63歳，男性．2004年10月に2週間続く食思不振，腹部膨満を主訴に来院し，著明な炎症反応を認めたため精査加療目的で入院した．既往に無治療の糖尿病があった．術前検査で4型胃癌と診断，生検結果は低分化型腺癌であった．開腹所見は胃全体の硬化とリンパ節腫大を認め，胃全摘・脾合併切除術を行った．病理組織学的所見では早期類似進行癌の他に，広範に炎症細胞浸潤と線維性変化を呈した化膿性胃炎があり，これが4型胃癌と誤認した原因であった．化膿性胃炎は稀で術前診断困難な疾患であり4型胃癌との鑑別がしばしば問題となる．胃癌の鑑別診断として本症を念頭に置く必要がある．

症例は60歳代の男性で, 2001年10月胃癌に対して幽門側胃切除術を施行されている. その際の病理組織学的検査所見は中分化型腺癌で深達度pSM, pN0でありstage Iaであった. その後, 外来にて経過観察中であったが, 2004年9月の上部消化管内視鏡検査にて, 残胃後壁に2型の腫瘍を認めた. 生検で低分化型腺癌の診断であったため, 手術目的で入院となった. 2004年9月残胃全摘術, 脾臓合併切除, 左副腎部分切除術を施行した. 術後病理組織学的検索では免疫染色でNSEおよびsynaptophisinが弱陽性, CAM5.2, CEAが陰性であった. また, 術前の精査にて他臓器に病変を認めないことから, 残胃原発の小細胞癌と診断した. 術後TS-1による化学療法を行っているが, 現時点で再発を認めていない. 残胃発生の小細胞癌の報告例は自験例を含めて6例のみとまれであるため報告した.

In the budding yeast Saccharomyces cerevisiae, microtubule-organizing centers called spindle pole bodies (SPBs) are embedded in the nuclear envelope, which remains intact throughout the cell cycle (closed mitosis). Kinetochores are tethered to SPBs by microtubules during most of the cell cycle, including G1 and M phases; however, it has been a topic of debate whether microtubule interaction is constantly maintained or transiently disrupted during chromosome duplication. Here, we show that centromeres are detached from microtubules for 1–2 min and displaced away from a spindle pole in early S phase. These detachment and displacement events are caused by centromere DNA replication, which results in disassembly of kinetochores. Soon afterward, kinetochores are reassembled, leading to their recapture by microtubules. We also show how kinetochores are subsequently transported poleward by microtubules. Our study gives new insights into kinetochore–microtubule interaction and kinetochore duplication during S phase in a closed mitosis.

In mitosis, kinetochores are initially captured by the lateral sides of single microtubules and are subsequently transported toward spindle poles. Mechanisms for kinetochore transport are not yet known. We present two mechanisms involved in microtubule-dependent poleward kinetochore transport in Saccharomyces cerevisiae. First, kinetochores slide along the microtubule lateral surface, which is mainly and probably exclusively driven by Kar3, a kinesin-14 family member that localizes at kinetochores. Second, kinetochores are tethered at the microtubule distal ends and pulled poleward as microtubules shrink (end-on pulling). Kinetochore sliding is often converted to end-on pulling, enabling more processive transport, but the opposite conversion is rare. The establishment of end-on pulling is partly hindered by Kar3, and its progression requires the Dam1 complex. We suggest that the Dam1 complexes, which probably encircle a single microtubule, can convert microtubule depolymerization into the poleward kinetochore-pulling force. Thus, microtubule-dependent poleward kinetochore transport is ensured by at least two distinct mechanisms.

This paper attempts to account for the changing distribution of lexical subjects in English infinitives within the framework of the Minimalist Program, paying special attention to the role of the infinitival morpheme and the change in the category and formal features of the infinitive marker to. First, it is argued that from Old English to the 16th century, when the infinitival morpheme was present, the external argument of bare infinitives could be realized either as a lexical DP or the infinitival morpheme; then, the loss of the infinitival morpheme led to the situation that bare infinitives obligatorily have lexical subjects, as we see in bare infinitive complements to causative verbs in Present-day English. Next, focusing change in the category and formal features of the infinitive marker to, the following development of to-infinitives is proposed: (i) in Old English, to as a preposition had an inherent Case feature licensing the infinitival morpheme, which in turn functioned as the external argument of to-infinitives; (ii) in Early Middle English, to came to have a structural Case feature with the optional EPP feature, giving rise to to-infinitives with lexical subjects that are licensed by to; (iii) in Late Middle English, to began to lose its structural Case feature and have the feature specification with the EPP feature only as a functional category, giving rise to to-infinitives with lexical subjects that are licensed by the matrix v, as in the case Present-day English; (iv) this specification became the only possibility after the loss of the infinitival morpheme in the 16th century, at least for the types of to-infinitives investigated here.

People with dementia often have low physical activity and some sleep problems. This study focused on daily life activities and sleeping conditions, and examined the use of these parameters for detecting dementia. Five passive infrared (IR) sensors were installed in each of 14 subject's houses. Each patient lived alone. The subjects' in-house movements were recorded by the passive IR sensor for approximately 3 months (average, 78 days). Based on these records, the following parameters of life activities were assessed: (1) the number of outings, (2) total sleep time, (3) number of sleep interruptions, and (4) sleep rhythm. Subjects with impaired cognition (Mini Mental State Examination [MMSE]24) had a significantly lesser number of outings (p = 0.001) and a tendency toward a shorter sleep time (p = 0.054) in comparison with control subjects (MMSEor= 24). These results suggest that the monitoring of life activities by using passive infrared sensors could be an efficient method for detecting dementia.

To examine and analyze the spread of measles in Osaka in 2014 and determine effective measures to prevent such occurrences.We analyzed 47 cases of measles reported in Osaka, including one measles patient living in another prefecture where there was an outbreak. We focused on age distribution, the number of patients reported each week, estimated infection routes, history of measles vaccination, detection of viruses, and number of days it took to report the case after the onset of measles.Patients aged 20-39 years accounted for 24 cases (51.1%). The number of patients reported started from 2nd week with relatively broad peak to 27(th) week, and the measles epidemic was brought under control in the 47(th) week. Among the 47 cases, no source could be identified in 16 cases (34.0%). Household exposure was the main cause of the infection (25.5%), followed by imported cases (21.3%). Eighty-three percent of the overall patients had not received a measles vaccination at all or it was unclear whether they previously had been vaccinated. Genotype B3, H1, and D8 were detected in our patients and these genotypes originated overseas. It took significantly more days, from the onset of measles, for the case to be reported in patients aged 15 years and over compared with those aged under 15 years (P=0.001).For eradicating measles in Osaka, it is important to raise awareness about this issue among medical institutions, especially institutions for adults, in order for them to report cases as soon as possible, upon discovery in their patients. In addition, "catch-up" supplementary immunizations are effective for all people, including adults who are susceptible to measles.

Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td = 137 °C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (ΔH) with no entropic gain from hydrophobic solvation around 100 °C and that the stabilization due to salt bridges resulted from both the increase in ΔH from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 °C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.

In order to develop novel anti-epileptic drugs that are effective for both general and partial seizure, we conducted in vivo screening of our chemical library in the mice MES and sc-PTZ models and found the benzothiazine 1 as lead compound. Optimization of this compound led to the discovery of compound 7b, which showed potent anticonvulsant effect in the MES, scPTZ and rat amygdala kindling models. Since the chemical structure of 7b is different from that of any existing AED, it is suggested that 7b may have unique mechanism of action for relieving both partial and generalized epilepsy.

The duration of viral shedding in the patients from two outbreaks and four sporadic cases of norovirus (NoV) infections was investigated. The longest period of viral shedding into feces was for 173 days in an inpatient from one case of outbreak. The VP1 sequence from two long-term viral shedding cases in the outbreak revealed four synonymous and one non-synonymous mutations in one inpatient at 26 days from the onset of illness, and nine synonymous and two non-synonymous mutations and a deletion, 10 synonymous mutations and a deletion in other inpatient at 29 days and 54 days from the onset of illness, respectively. Ten of the 11 amino acid positions detected in these two inpatients were in the outermost P2 domain of the viral capsid protein, and mutations at positions 295, 297, and 394 were shared in the inpatients. Mutations in the P2 domain were in epitopes A and D or near epitopes A, C, and E, suggesting that the long-term carrier state of norovirus infection contributes to the generation of escape mutants by host immunoselection.

Noroviruses (NoVs) commonly cause acute gastroenteritis outbreaks. Broadly reactive diagnostic assays are essential for rapid detection of NoV infections. We previously generated a panel of broadly reactive monoclonal antibodies (MAbs). We characterized MAb reactivities by use of virus-like particles (VLPs) from 16 different NoV genotypes (6 from genogroup I [GI], 9 from GII, and 1 from GIV) coating a microtiter plate (direct enzyme-linked immunosorbent assay [ELISA]) and by Western blotting. MAbs were genotype specific or recognized multiple genotypes within a genogroup and between genogroups. We next applied surface plasmon resonance (SPR) analysis to measure MAb dissociation constants (Kd) as a surrogate for binding affinity; aKdlevel of Kdcould not be measured by SPR analysis also failed to capture the NoV VLPs; in contrast, those with a measurableKdgave a positive signal in the capture ELISA. Thus, some broadly cross-reactive epitopes in the VP1 protruding domain may be partially masked on intact particles. One MAb, NV23, was able to detect genogroup I, II, and IV VLPs from 16 genotypes tested by sandwich ELISA, and it successfully detected NoVs in stool samples positive by real-time reverse transcription-PCR when the threshold cycle (CT) value was

Monoclonal antibodies (MAbs) are important for in-depth antigenic characterization and diagnosis of infections with human caliciviruses that cause almost all outbreaks of nonbacterial gastroenteritis. We compared different routes of immunization with nonreplicating virus-like particles (VLPs) from recombinant Norwalk virus (rNV) and recombinant Mexico virus (rMX) administered to BALB/c mice to determine the efficiency of hybridoma production. Oral immunization with VLPs without adjuvant resulted in high yields of MAb-secreting hybridomas (90%) to these VLPs of IgG (61%), IgM (29%) and IgA (10%) isotypes. Fusions with mesenteric lymph node lymphocytes yielded MAbs of various subclasses including IgG2a, IgG3, IgM and IgA. These results suggest that an immunization route that mimics the natural route of viral infection pathway may facilitate MAb technology by increasing the yields of antibody secreting hybridoma cells.

Human norovirus (NoV) strains cause a considerable number of outbreaks of gastroenteritis worldwide. Based on their capsid gene (VP1) sequence, human NoV strains can be grouped into two genogroups (GI and GII) and at least 14 GI and 17 GII genotypes (GI/1–14 and GII/1–17). Human NoV strains cannot be propagated in cell-culture systems, but expression of recombinant VP1 in insect cells results in the formation of virus-like particles (VLPs). In order to understand NoV antigenic relationships better, cross-reactivity among 26 different NoV VLPs was analysed. Phylogenetic analyses grouped these NoV strains into six GI and 12 GII genotypes. An antibody ELISA using polyclonal antisera raised against these VLPs was used to determine cross-reactivity. Antisera reacted strongly with homologous VLPs; however, a number of novel cross-reactivities among different genotypes was observed. For example, GI/11 antiserum showed a broad-range cross-reactivity, detecting two GI and 10 GII genotypes. Likewise, GII/1, GII/10 and GII/12 antisera showed a broad-range cross-reactivity, detecting several other distinct GII genotypes. Alignment of VP1 amino acid sequences suggested that these broad-range cross-reactivities were due to conserved amino acid residues located within the shell and/or P1-1 domains. However, unusual cross-reactivities among different GII/3 antisera were found, with the results indicating that both conserved amino acid residues and VP1 secondary structures influence antigenicity.

A one-dimensional gas hydrate accumulation model is developed to simulate the existence of hydrate in the Nankai Trough margin, where the total organic carbon content is extremely low. In order to study the accumulation of gas hydrate along a seismic profile, our model assumes an initial 1000 m thick turbidite sedimentation at the trench axis, and then the sediment experiences tectonic uplift to form the Nankai accretionary prism. Methane generated by microbial processes during the sedimentation is partly trapped in the pore space and partly migrated upwards due to compaction and subsequent porosity decrease at deeper levels. Upward methane gas flux from the deeper part (below 1000 m) is predicted by the model because the maximum thickness of the accreted sediment exceeds ten kilometers in the Nankai Trough accretionary prism. The basic geological parameters input to the model are those obtained from the ODP site 808, which was drilled at the lower inner trench slope. The methane hydrate stability zone is essentially controlled by bottom water temperature, regional heat flow and hydrostatic pressure. The simulation shows that methane hydrate will not accumulate in the Nankai Trough margin if only microbial methane generation is considered because the organic carbon content in the Nankai Trough area is as low as 0.75%. However, if we assume an upward flux of 5 kg of methane per square meters per 10,000 years, it will cause approximately 10 to 25% hydrate saturation under current physical conditions. The origin of the methane may be thermogenic and/or microbial. The resultant distribution of methane hydrate shows very good correlation with the distribution of the BSR.

During the cell-division cycle, chromosomal DNA must initially be precisely duplicated and then correctly segregated to daughter cells. The accuracy of these two events is maintained by two interlinked cycles: the replication licensing cycle, which ensures precise duplication of DNA, and the cohesion cycle, which ensures correct segregation. Here we provide a general overview of how these two systems are coordinated to maintain genetic stability during the cell cycle.

For proper chromosome segregation, sister kinetochores must attach to microtubules extending from opposite spindle poles prior to anaphase onset. This state is called sister kinetochore bi-orientation or chromosome bi-orientation. The mechanism ensuring chromosome bi-orientation lies at the heart of chromosome segregation, but is still poorly understood. Recent evidence suggests that mal-oriented kinetochore-to-pole connections are corrected in a tension-dependent mechanism. The cohesin complex and the Ipl1/Aurora B protein kinase seem to be key regulators for this correction. In this article, I discuss how cells ensure sister kinetochore bi-orientation for all chromosomes, mainly focusing on our recent findings in budding yeast.

Immunotherapy against a variety of malignancies, including pleural-based malignancies, has shown promise in animal models and early human clinical trials, but successful efforts will need to address immunosuppressive factors of the tumor and host, particularly certain cytokines and CD4(+) CD25(+) regulatory T cells (Treg). Here, we evaluated the cellular and cytokine components of malignant pleural effusions from 44 patients with previously diagnosed mesothelioma, non-small cell lung cancer (NSCLC), or breast cancer and found significant differences in the immune profile of pleural effusions secondary to mesothelioma vs. carcinoma. Although a high prevalence of functionally suppressive CD4(+) CD25(+) T cells was found in carcinomatous pleural effusions, mesothelioma pleural effusions contained significantly fewer CD4(+) CD25(+) T cells. Activated CD8(+) T cells in pleural fluid were significantly more prevalent in mesothelioma than carcinoma. However, there is clear patient-to-patient variability and occasional mesothelioma patients with high percentages of CD4(+) CD25(+) pleural effusion T cells and low percentages of CD8(+) CD25(+) pleural effusion T cells can be identified. Mesothelioma pleural effusions contained the highest concentrations of the immunosuppressive cytokine transforming growth factor (TGF)-beta. Thus, the contribution of cellular and cytokine components of immunosuppression associated with malignant pleural effusions varies by tumor histology and by the individual patient. These results have implications for the development of immunotherapy directed to the malignant pleural space, and suggest the need to tailor immunotherapy to overcome immunosuppressive mechanisms in tumor environments.

Lung cancer is the leading cause of cancer death with non–small-cell lung cancer (NSCLC) accounting for approximately 80% of thoracic malignancies. The overall survival for NSCLC remains poor, with only 12% to 14% of patients surviving 5 years from diagnosis.1 Even in those patients who are eligible for surgery, more than one quarter of cases relapse, usually at distant sites.2,3 Novel approaches to the management of lung cancer are urgently required. One potential therapeutic target in cyclooxygenase- (COX) 2. COX is a key enzyme in the conversion of arachidonic acid to prostaglandins (PGs) and other eicosanoids. COX-1 is a housekeeping gene expressed in most tissues. COX-2 usually is absent but is induced by inflammatory and mitogenic stimuli, resulting in increased synthesis of PGs in inflamed and neoplastic tissues. COX-2 mRNA and protein levels are overexpressed in several types of human malignancies,4,5 including lung cancer6, suggesting that COX-2 is mechanistically linked to the development of cancer. Clinical data in support of this hypothesis are recent studies showing that COX-2 overexpression is a marker of poor prognosis in stage I NSCLC7,8 and is also associated with invasion and metastasis in lung cancer.9,10 Experimental data have also been generated suggesting an important role of COX-2 overexpression in lung cancer pathogenesis. Several studies have demonstrated that selective COX-2 inhibitors could inhibit the in vitro growth of human and mouse lung cancer cell lines.11–13 The use of COX-2 inhibitors in combination with conventional anticancer agents has also demonstrated additive antigrowth effects in human lung cancer cell lines.14,15 The antitumor activities of COX-2 inhibitors have also been studied in selected mouse models of NSCLC. Administration of the selective COX-2 inhibitors celecoxib and SC-58263 resulted in decreased tumor growth of the Lewis lung carcinoma (LLC) line in C57/B6 mice.16–18 The COX-2 inhibitor JTE-522 decreased growth of a human lung cancer line in nude mice.15 Two chemoprevention studies in mice have been reported. Rioux and Castonguay19 showed prevention of NNK-induced lung tumorigenesis in A/J mice using the selective COX-2 inhibitor NS-398. In contrast, Kisley et al20 reported that celecoxib reduced inflammation but not tumorigenesis in mice in a protocol in which methylcholanthrene was followed by chronic butylated hydroxytoluene. Key limitations of the treatment studies mentioned above were that only very limited number of models were tested (ie, only the nonimmunogenic LLC mouse lung cancer cell line) and that very high doses of COX-2 inhibitors were used, doses that would be difficult to achieve in human patients. The goal of this study was to evaluate the value and limitations of a specific COX-2 inhibitor, rofecoxib, using an oral dosing regimen that resulted in serum levels that were similar to those that could be achieved in a clinical trial. Because augmentation of antitumor immune responses is one of the key antitumor mechanisms through which COX-2 inhibition may work (21; also see Discussion), we felt it important to test oral rofecoxib in mouse lung cancer models with different levels of immunogenicity. Based on our findings that COX-2 inhibition was only effective in small tumors, we also investigated the potential value of rofecoxib as adjuvant therapy after debulking surgery.

A total of 103 methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients and 78 MRSA from hospital workers during the years 1990 to 1994, and 52 MRSA from patients in year 2000, in one hospital at Wakayama Prefecture, were typed by pulsed-field gel electrophoresis (PFGE) profiles. MRSA isolates were grouped into 20 genomic types. These types were further divided into 57 subtypes. The type 1 was dominant among patients, and closely related type 1 strains were spread during 1990-1993. In contrast, the type 2 was dominant among medical workers in the same period. In 2000, the type 2 and 4 strains increased, and the diversity and complication of types appeared. The common types between the patients and medical workers were only type 1 to 3. Some strains isolated from both during the same period were shown to have the same types. Others were shown to be patient-specific types. These results suggest that there are complicated transmissions of MRSA in the hospital, i.e., the endogenous infection, patient-patient cross infection and medical worker-patient cross infection.