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Round spermatid injection (ROSI) into unfertilized oocytes enables a male with a severe spermatogenesis disorder to have children. One limitation of the application of this technique in the clinic is the identification and isolation of round spermatids from testis tissue. Here we developed an efficient and simple method to isolate rodent haploid round spermatids using flow cytometric cell sorting, based on DNA content (stained with Hoechst 33342 or Dye Cycle Violet) or by cell diameter and granularity (forward and side scatter). ROSI was performed with round spermatids selected by flow cytometry, and we obtained healthy offspring from unstained cells. This non-invasive method could therefore be an effective option for breeding domestic animals and human male infertility treatment. Mol. Reprod. Dev. 83: 488-496, 2016. © 2016 Wiley Periodicals, Inc.

Introduction As a component of midwife care, eliminating clothing that constricts the trunk has been shown to markedly elevate the uterine fundus, soften the uterus and abdomen, and reduce the abdominal wall tension in women admitted to hospital due to the risk of miscarriage or premature delivery. However, no prospective study has conclusively verified the efficacy of avoiding constrictive clothes around the trunk in pregnant women. We aim to verify the efficacy of instructing pregnant women to wear loose clothing that does not constrict the trunk to reduce the risk of premature birth and improve quality of life (QoL) during pregnancy. Methods and analysis We will conduct a randomised controlled trial of pregnant women scheduled to deliver at the National Center for Child Health and Development in Tokyo, Japan. A total of 616 pregnant women, from whom written informed consent will be obtained, will be allocated randomly to an intervention group or a control group. Women in the control group will be provided with anaemia prevention leaflets at 20 weeks’ gestation and skin-care leaflets at 30 weeks’ gestation. Women in the intervention group will be provided with the same leaflets and will also receive health advice from health professionals to avoid constrictive clothing around the trunk. The primary outcome will be a difference between these groups in the frequency of any one of the following category variables: (1) cervical length

The aim of this article is to introduce the proteomic analysis of psychiatric disorders and psychological stress and the ongoing study on Tsunami victims in Srilanka.

スターバー抽出（SBSE）法を試料前処理に用い，高速液体クロマトグラフィー－蛍光検出法（HPLC-FL）により，河川水中の多環芳香族炭化水素（PAHs）の分析法を構築した．測定対象物質として，フルオランテン（Fl），ベンゾ[b]フルオランテン（B[b]F），ベンゾ[k]フルオランテン（B[k]F），ベンゾ[a]ピレン（B[a]P）を選定した．河川水（100 ml）にメタノール（10 ml）及びポリジメチルシロキサン（PDMS）かくはん子を加え，スターラー（800 rpm）で3時間かくはんした．その後，PDMSかくはん子を取り出し，アセトニトリル200 μlで溶媒脱着を行い，その10 μlをHPLC-FLにより分析した．本法の検出限界及び定量限界は，それぞれ，0.6～6.0 pg/ml及び2.0～20 pg/mlとなり，高感度であった．添加回収試験の結果，回収率94.8％ 以上と良好であった．本法を用いて，多摩川から採取した河川水を測定した結果，すべての検体からFl（10～19 pg/ml）及びB[a]P（32～52 pg/ml）が検出された．

We have developed a molecularly imprinted polymer (MIP) using a stable isotope labeled compound as the template molecule and called it the “isotope molecularly imprinted polymer” (IMIP). In this study, bisphenol A (BPA) was used as the model compound. None imprinted polymer (NIP), MIP, dummy molecularly imprinted polymer (DMIP) and IMIP were prepared by the suspension polymerization method using without template, BPA, 4-tert-butylphenol (BP) and bisphenol A-d16 (BPA-d16), respectively. The polymers were subjected to molecularly imprinted solid phase extraction (MI-SPE), and the extracted samples were subjected to liquid chromatography–mass spectrometry (LC–MS). Although the leakage of BPA-d16 from the IMIP was observed and that of BPA was not observed. The selectivity factors of MIP and IMIP for BPA were 4.45 and 4.43, respectively. Therefore, IMIP had the same molecular recognition ability as MIP. When MI-SPE with IMIP was used and followed by LC–MS in the analysis of river water sample, the detection limit of BPA was 1 ppt with high sensitivity. Moreover, the average recovery was higher than 99.8% (R.S.D.: 3.7%) by using bisphenol A-13C12 (BPA-13C12) as the surrogate standard. In addition, the IMIP were employed in MI-SPE of BPA in river water sample by LC–MS. The concentration of BPA in the river water sample was determined to be 32 pg ml−1. We confirmed that it was possible to measure trace amounts of a target analyte by MI-SPE using IMIP.

A new method, stir bar sorptive extraction (SBSE) with in situ derivatization and thermal desorption (TD)–gas chromatography–mass spectrometry (GC–MS), which is used for the determination of trace amounts of chlorophenols, such as 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TrCP), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP) and pentachlorophenol (PCP), in tap water, river water and human urine samples, is described. The derivatization conditions with acetic acid anhydride and the SBSE conditions such as extraction time are investigated. Then, the stir bar is subjected to TD followed by GC–MS. The detection limits of the chlorophenols in tap water, river water and human urine samples are 1–2, 1–2, and 10–20 pg ml−1 (ppt), respectively. The calibration curves for the chlorophenols are linear and have correlation coefficients higher than 0.99. The average recoveries of the chlorophenols in all the samples are higher than 95% (R.S.D.

A novel method for the trace analysis of natural and synthetics estrogens, such as estrone (E1), 17beta-estradiol (E2) and 17alpha-ethynylestradiol (EE), in river water sample was developed, which involved stir bar sorptive extraction (SBSE) with in situ derivatization followed by thermal desorption (TD)-gas chromatography-mass spectrometry (GC-MS). The derivatization conditions with acetic acid anhydride and the SBSE conditions such as sample volume and extraction time were investigated. In addition, the single and multi-shot modes in TD were investigated. The detection limits of E1, E2 and EE in river water sample were 0.2, 0.5 and 1 pg ml(-1) (ppt), respectively, in the multi-shot mode using five stir bars. The calibration curves for E1, E2 and EE were linear and had correlation coefficients >0.99. The average recoveries of E1, E2 and EE from all sample volumes were higher than 90% (R.S.D. < 10%) with correction using an added surrogate standard such as estrone-13C4, 17beta-estradiol-13C4 or 17alpha-ethynylestradiol-13C4. This simple, accurate, sensitive and selective analytical method may be applicable to the determination of trace amounts of estrogens in water samples.

Introduction The study investigated the effectiveness of Shiatsu therapy in relation to the management of health and wellbeing concerns of cancer and palliative care patients in an out-patient clinic. Method Patients are referred to the Complementary Therapies Service for symptom management, particularly stress and anxiety, but also other symptoms such as nausea or insomnia. Data was collected following use of the Measure Yourself Concerns and Wellbeing (MYCaW) questionnaire, which was designed for evaluating supportive care interventions. Results Mean changes in post-intervention MYCaW scores were highly significant (p Conclusion Anxiety, stress management and pain scores were the most improved. Wellbeing scores also improved, on average, by two points on the Likert scale. Patients stated that ‘being listened to’ and ‘being heard’ were important factors when describing how Shiatsu had helped. We suggest that a study using larger numbers is necessary in order to provide more robust evidence rather than emerging trends.

To understand radiocesium transfer in the forest insect food web, we investigated the activity concentrations of radiocesium in forest insects in the Fukushima and Ibaraki Prefectures approximately 1.5-2.5 years after the Fukushima Dai-ichi Nuclear Power Plant. We analyzed 34 species of insects sampled from 4 orders and 4 feeding functional groups (herbivore, carnivore, omnivore, and detritivore) from three sites in each prefecture. 137Cs activity concentrations were lowest in herbivorous species and were especially high in detritivorous and omnivorous species that feed on forest litter and fungi. Radiocesium activity concentrations in any given species reflected the degree of contamination of that species' primary food sources since radiocesium activity concentrations were found to be the lowest in leaves and grass and the highest in litter, bark, and fungi. This study confirmed that litter and other highly contaminated forest components such as fungi, decaying wood, bryophytes, and lichens serve as sources of 137Cs transfer into the forest insect community.

While the role of p75(NTR) signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75(NTR) on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75(NTR)-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75(NTR) in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75(NTR), as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75(NTR)-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75(NTR) signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.

Cytotoxic T-lymphocytes (CTLs) specific for tumor-associated antigens (TAAs) act in the immune surveillance system as major effector cells to eliminate malignant cells. Immunization with TAA-loaded dendritic cells (DCs) has great potential for treating cancer, because DCs are potent antigen-presenting cells capable of inducing antigen-specific CTLs by the primary activation of naive T-lymphocytes. The establishment of a non-cytotoxic and efficient antigen delivery method is required to improve the efficacy of DC-based cancer immunotherapy. We developed biodegradable poly(γ-glutamic acid) nanoparticles (γ-PGA NPs) that can efficiently entrap various proteins as antigen delivery carriers. γ-PGA NPs efficiently delivered entrapped antigenic proteins into DCs without cytotoxicity and presented antigens to DCs via major histocompatibility complex class I and II molecules. Immunization with TAA-loaded DCs using γ-PGA NPs inhibited tumor growth by inducing TAA-specific CTLs. These findings indicate that γ-PGA NPs can function as useful antigen delivery carriers in DC-based cancer immunotherapy.

Although disturbed cytokinesis of mononuclear Hodgkin (H) cells is thought to generate Reed-Sternberg (RS) cells, differentiation of Hodgkin's lymphoma (HL) cells is not fully understood. Recent studies indicate that cells found in a side population (SP) share characteristics of cancer stem cells. In this study we identified an SP in the HL cell lines, KMH2 and L428. This SP almost entirely consists of distinct small mononuclear cells, whereas the non-SP is a mixture of relatively large cells with H or RS cell-like morphology. Culture of the small mononuclear cells in the SP from KMH2 generated a non-SP. Single cell culture of the SP cells generated large cells with H or RS cell-like morphology. We found that CD30 overexpression and constitutive nuclear factor-κB (NF-κB) activity, both of which are characteristics of HL cells, are shared between the SP and non-SP cells for both KMH2 and L428. Inhibition of NF-κB induced apoptosis in both fractions, whereas the SP cells were resistant to a conventional chemotherapeutic agent doxorubicin. The results show that HL cell lines contain an SP, that is enriched for distinct small mononuclear cells and generates larger cells with H and RS cell-like morphology. The results also stress the significance of NF-κB inhibition for eradication of HL cells.

Recent studies have demonstrated that human dental pulp contains adult stem cells. A pulse of the thymidine analog BrdU given to young animals at the optimal time could clarify where slow-cycling long-term label-retaining cells (LRCs), putative adult stem cells, reside in the pulp tissue. This study focuses on the mapping of LRCs in growing teeth and their regenerative capacity after tooth injuries. Two to seven peritoneal injections of BrdU into pregnant Wistar rats revealed slow-cycling long-term dense LRCs in the mature tissues of born animals. Numerous dense LRCs were postnatally decreased in number and reached a plateau at 4 weeks after birth when they mainly resided in the center of the dental pulp, associating with blood vessels. Mature dental pulp cells were stained with Hoechst 33342 and sorted into (

In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in Gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but not ADP by a mechanism independent of ATP hydrolysis. Unlike E. coli DnaA, binding of ADP to S. aureus DnaA was unstable. As a result, at physiological concentrations of ATP, ADP bound to S. aureus DnaA was rapidly exchanged for ATP, thereby regenerating the ability of DnaA to form the open complex in vitro. Therefore, we examined whether formation of ADP-DnaA participates in suppression of replication initiation in vivo. Induction of the R318H mutant of the AAA+ sensor 2 protein, which has decreased intrinsic ATPase activity, caused over-initiation of chromosome replication in S. aureus, suggesting that formation of ADP-DnaA suppresses the initiation step in S. aureus. Together with the biochemical features of S. aureus DnaA, the weak ability to convert ATP-DnaA into ADP-DnaA and the instability of ADP-DnaA, these results suggest that there may be unidentified system(s) for reducing the cellular ratio of ATP-DnaA to ADP-DnaA in S. aureus and thereby delaying the re-initiation of DNA replication.

Although human bone marrow stromal cells (BMSCs) have the ability to form bone when transplanted, the responsible factors for in vivo osteogenic abilities are poorly understood. Here we report conditions that are required for human BMSCs to demonstrate their in vivo osteogenic abilities. BMSCs were obtained from healthy donors and their in vivo osteogenic abilities were analyzed. Transplantation analyses revealed that the passage number and length of osteogenic induction significantly affected ectopic bone formation. Although 2-week induction increased the percentage of success in bone formation compared with the 1-week induction, BMSCs completely lost their in vivo osteogenic ability after passage 4 regardless of the length of osteogenic induction. Despite their in vivo osteogenic ability, no significant difference was observed in alkaline phosphatase activity or gene expression of osteogenic markers between BMSCs at passages 1 and 3. Differences were only observed in in vitro mineralizing abilities. Application of basic fibroblast growth factor helped to maintain the BMSCs in vivo osteogenic ability; basic fibroblast growth factor altered cell growth and expression of HLA-DR. The results strongly suggest that there are several required conditions for human BMSCs to demonstrate their bone-forming capabilities, which should be further investigated and considered when designing a protocol for clinical bone tissue engineering.

Current standard techniques for bone tissue engineering utilize ex vivo expanded osteogenic cells. However, ex vivo expansion requires serum, which may hinder clinical applications. Here, we report the feasibility and efficacy of bone tissue engineering with human bone marrow stromal cells (BMSCs) expanded in serum-free conditions. Bone marrow was aspirated from 4 healthy donors and adherent cells were cultured in either serum-free medium (STEMPRO((R)) MSC SFM) or conventional serum-containing medium (alpha-MEM supplemented with 10% serum). Efficacy of expansion was greater in serum-free medium. Phenotypically, serum-free expanded BMSCs were smaller in cell-size and showed expression of CD105(++) and CD146(dim). After osteogenic induction, serum-free expanded BMSCs showed lower alkaline phosphatase activity. However, they showed higher responsiveness to induction. In vivo bone-forming ability was also confirmed. In conclusion, bone tissue engineering with serum-free expanded BMSCs is feasible and as efficient as that obtained with BMSCs expanded in conventional serum-containing medium.

That generation of allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies (ASHmAb) is very difficult is well known. This is thought to be due to the unique epitope structure, an assemblage of amino acid residues that lie separately in the amino acid sequence of human HLA, and to its low antigenicity compared with that of common epitopes recognized as xenogeneic determinants by mice. Here we report a rapid and efficient strategy to generate ASHmAb. Different from usual immunization methods is that we suppressed the production of non-allele-specific anti-HLA antibodies against xenogeneic determinants of HLA molecules by immunizing human HLA-B51 transgenic mice against non-HLA-B51 HLA tetramers. In addition, HLA-coated beads enabled rapid and efficient screening for ASHmAb. ASHmAb generated by this strategy will be useful for HLA typing and for clinical diagnosis, such as flow cytometry-based chimerism analysis for early detection of graft failure and relapse of leukemia after HLA-mismatched hematopoietic stem cell transplantation.

One of the most important anthropic missions is preventing the global spread of infectious diseases. Vaccination is the only available preventive treatment for infectious diseases, but the availability of vaccines in developing countries is not adequate. We report a simple, easy-to-use, noninvasive hydrogel patch transcutaneous vaccination system. Antigen (Ag)-specific IgG production was induced by applying an Ag-immersed patch to non-pretreated mouse auricle or hairless rat back skin. Immunofluorescence histochemical analysis revealed that Langerhans cells resident in the epidermal layer captured the antigenic proteins delivered by the hydrogel patch, which promoted the penetration of antigenic proteins through the stratum corneum, and that Ag-capturing Langerhans cells migrated into draining lymph nodes. Humoral immunity elicited by our transcutaneous vaccination system demonstrated neutralizing activity in both adenoviral infection and passive-challenge tetanus toxin experiments. The use of this hydrogel patch transcutaneous vaccination system will facilitate the global distribution of effective and convenient vaccines.

Introduction As a component of midwife care, eliminating clothing that constricts the trunk has been shown to markedly elevate the uterine fundus, soften the uterus and abdomen, and reduce the abdominal wall tension in women admitted to hospital due to the risk of miscarriage or premature delivery. However, no prospective study has conclusively verified the efficacy of avoiding constrictive clothes around the trunk in pregnant women. We aim to verify the efficacy of instructing pregnant women to wear loose clothing that does not constrict the trunk to reduce the risk of premature birth and improve quality of life (QoL) during pregnancy. Methods and analysis We will conduct a randomised controlled trial of pregnant women scheduled to deliver at the National Center for Child Health and Development in Tokyo, Japan. A total of 616 pregnant women, from whom written informed consent will be obtained, will be allocated randomly to an intervention group or a control group. Women in the control group will be provided with anaemia prevention leaflets at 20 weeks’ gestation and skin-care leaflets at 30 weeks’ gestation. Women in the intervention group will be provided with the same leaflets and will also receive health advice from health professionals to avoid constrictive clothing around the trunk. The primary outcome will be a difference between these groups in the frequency of any one of the following category variables: (1) cervical length Ethics and dissemination The Institutional Review Board and Ethics Committee at the National Center for Child Health and Development, Japan, has approved this study. Our findings will be widely disseminated through conference presentations and peer-reviewed publications. Trial registration number UMIN000016853.

Some ergot alkaloids relaxed catch contraction of an isolated molluscan smooth muscle (anterior byssus retractor muscle of Mytilus edulis). Haloperidol, a competitive dopamine antagonist, shifted the dose response curves of ergot alkaloids, but methysergide did not. Bromocriptine, a potent dopaminergic ergot alkaloid, did not relax catch contraction. These results suggest that relaxation of catch contraction by some ergot alkaloids is mediated through dopamine receptors of this muscle, but dopamine receptors of this muscle seem to be somewhat different from those of mammalian brain.