Advanced Interferometry with 3-D Structured Illumination Reveals the Surface Fine Structure of Complex Biospecimens.

Interference reflection microscopy (IRM) is a powerful, label-free technique to visualize the surface structure of biospecimens. However, stray light outside a focal plane obscures the surface fine structures beyond the diffraction limit (dxy ≈ 200 nm). Here, we developed an advanced interferometry approach to visualize the surface fine structure of complex biospecimens, ranging from protein assemblies to single cells. Compared to 2-D, our unique 3-D structure illumination introduced to IRM enabled successful visualization of fine structures and the dynamics of protein crystal growth under lateral (dx-y ≈ 110 nm) and axial (dx-z ≤ 5 nm) resolutions and dynamical adhesion of microtubule fiber networks with lateral resolution (dx-y ≈ 120 nm), 10 times greater than unstructured IRM (dx-y ≈ 1000 nm). Simultaneous reflection/fluorescence imaging provides new physical fingerprints for studying complex biospecimens and biological processes such as myogenic differentiation and highlights the potential use of advanced interferometry to study key nanostructures of complex biospecimens.

Paternal involvement in infant care and developmental milestone outcomes at age 3 years: the Japan Environment and Children's Study (JECS).

BACKGROUND: In developed countries, the time fathers spend on childcare has increased steadily in recent decades. However, studies on the relationship between paternal care and child outcomes remain scarce. Thus, we examined the association between paternal involvement in childcare and children's developmental outcomes. METHODS: We used Japan's largest birth cohort data, the Japan Environment and Children's Study, to examine the relationship between paternal involvement in childcare at the child's age of 6 months and developmental milestone outcomes at the child's age of 3 years (n = 28,050). Developmental delays were assessed with Ages and Stages Questionnaire. Potential mediation by maternal parenting stress at the child's age of 1.5 years was also examined. We used log-binomial regression analyses to estimate risk ratios. RESULTS: Fathers' high involvement in childcare was associated with a lower risk of developmental delay in gross-motor, fine-motor, problem solving, and personal-social domains compared with low involvement, adjusting for potential confounders. For example, the risk ratio with 95% confidence intervals was 0.76 [0.67, 0.86] for the gross-motor domain. We also observed that the associations were partially mediated by maternal parenting stress. CONCLUSIONS: Fathers' active involvement in childcare during infancy may promote young children's development, partially by reducing maternal parenting stress. IMPACT: Using Japan's largest birth cohort data (Japan Environment and Children's Study), we showed that paternal involvement in infant care might benefit young children's development. Fathers' active involvement in infant care was associated with a lower risk of developmental delays in gross-motor, fine-motor, problem solving, and personal-social domains. Maternal parenting stress may mediate the association between paternal involvement in infant care and child development outcomes at 3 years.

Construction of a T7 phage random peptide library by combining seamless cloning with in vitro translation.

T7 phage libraries displaying random peptides are powerful tools for screening peptide sequences that bind to various target molecules. The T7 phage system has the advantage of less biased peptide distribution compared to the M13 phage system. However, the construction of T7 phage DNA is challenging due to its long 36 kb linear DNA. Furthermore, the diversity of the libraries depends strongly on the efficiency of commercially available packaging extracts. To address these issues, we examined the combination of seamless cloning with cell-free translation systems. Seamless cloning technologies have been widely used to construct short circular plasmid DNA, and several recent studies showed that cell-free translation can achieve more diverse phage packaging. In this study, we combined these techniques to construct four libraries (CX7C, CX9C, CX11C and CX13C) with different random regions lengths. The libraries thus obtained all showed diversity > 109 plaque forming units (pfu). Evaluating our libraries with an anti-FLAG monoclonal antibody yielded the correct epitope sequence. The results indicate that our libraries are useful for screening peptide epitopes against antibodies. These findings suggest that our system can efficiently construct T7 phage libraries with greater diversity than previous systems.

Efficient derivation and banking of clinical-grade human embryonic stem cell lines in accordance with Japanese regulations.

Introduction: We recently established clinical-grade human embryonic stem cell (hESC) line KthES11 in accordance with current good manufacturing practice standards in Japan. Despite this success, the establishment efficiency was very low at 7.1% in the first period. Methods: To establish clinical-grade hESC lines, we used xeno-free chemically defined medium StemFit AK03N with the LM-E8 fragments instead of feeder cells. The protocol was then optimized, especially in the early culture phase. Results: We established five hESC lines (KthES12, KthES13, KthES14, KthES15, and KthES16) with 45.5% efficiency. All five hESC lines showed typical hESC-like morphology, a normal karyotype, pluripotent state, and differentiation potential for all three germ layers. Furthermore, we developed efficient procedures to prepare master cell stocks for clinical-grade hESC lines and an efficient strategy for quality control testing. Conclusions: Our master cell stocks of hESC lines may contribute to therapeutic applications using human pluripotent stem cells in Japan and other countries.

Low Surface Potential with Glycoconjugates Determines Insect Cell Adhesion at Room Temperature.

Cell-coupled field-effect transistor (FET) biosensors have attracted considerable attention because of their high sensitivity to biomolecules. The use of insect cells (Sf21) as a core sensor element is advantageous due to their stable adhesion to sensors at room temperature. Although visualization of the insect cell-substrate interface leads to logical amplification of signals, the spatiotemporal processes at the interfaces have not yet been elucidated. We quantitatively monitored the adhesion dynamics of Sf21 using interference reflection microscopy (IRM). Specific adhesion signatures with ring-like patches along the cellular periphery were detected. A combination of zeta potential measurements and lectin staining identified specific glycoconjugates with low electrostatic potentials. The ring-like structures were disrupted after cholesterol depletion, suggesting a raft domain along the cell periphery. Our results indicate dynamic and asymmetric cell adhesion is due to low electrostatic repulsion with fluidic sugar rafts. We envision the logical design of cell-sensor interfaces with an electrical model that accounts for actual adhesion interfaces.

Definitive Endoderm from EpiSC Aggregates in Matrigel.

This chapter describes the protocol to derive definitive endoderm cells from epiblast stem cells (EpiSCs) via a process analogous to gastrulation in embryos. The basis of this protocol mimicking the in vivo gastrulation process makes a contrast with those using sequential administration of pharmacological molecules and recombinant signaling proteins even at nonphysiological levels. In the experimental setup, EpiSCs are first freed from the dish-adherent condition to form free-floating aggregates, where endoderm precursor pools are produced. Embedding the EpiSC aggregates in the Matrigel allows the endoderm precursors to interact with the Matrigel mimicking the laminin-rich basement membrane underlying the egg cylinder epiblast in embryos, and let the precursors migrate into the Matrigel-filled external zone and develop into endodermal epithelial tissues.

Nap patterns of children in kindergartens and childcare transit facility: a study in northern Peninsular Malaysia.

Objective: The purpose of this study was to compare the duration of sleep and nap patterns between children in private kindergartens and those in a tabika-transit facility, in Malaysia. Methods: The sleep duration of children aged 3-6, who attended either a kindergarten or a childcare transit facility (tabika-transit in Malay) was investigated. Observational sleeping records were maintained for 24 hours, over 14 consecutive days. A self-administered questionnaire for caregivers investigated the children's lifestyles. Naps were optional at kindergartens but mandatory at the tabika-transit. Results: Of 35 participating children, data from 33 were analyzed. All respondents belonged to the Malay ethnicity, with 16 boys (48.5%) and 17 girls (51.5%). The average age of the children was 5.4 years; 11 of them were from kindergartens and 22 from a tabika-transit. The children slept longer and woke up later on weekends than on weekdays. There was a significant difference in the naptaking rate between the two groups; it was 100% in the tabika-transit, and 30% in the kindergartens during weekdays. However, on weekends, 19 of 22 tabika- transit children did not nap on any of the days (86.4%). The kindergarten group's naps showed no outstanding differences between weekdays and weekends. Concerning the bedtime and wake-up times, no differences were found between the two groups. Discussion: During weekdays, all children in the tabika-transit took naps, whereas one-third of them did in the kindergartens. Larger study is needed to assess how this mandatory napping style affects children's lifestyle and development.

Modeling early stages of endoderm development in epiblast stem cell aggregates with supply of extracellular matrices.

Endoderm precursors expressing FoxA2 and Sox17 develop from the epiblast through the gastrulation process. In this study, we developed an experimental system to model the endoderm-generating gastrulation process using epiblast stem cells (EpiSCs). To this end, we established an EpiSC line i22, in which enhanced green fluorescent protein is coexpressed with Foxa2. Culturing i22 EpiSCs as aggregates for a few days was sufficient to initiate Foxa2 expression, and further culturing of the aggregates in Matrigel promoted the sequential activation of transcription factor genes involved in endoderm precursor development, e.g., Eomes, Gsc, and Sox17. In aggregation culture of i22 cells for 3 days, all cells expressed POU5F1, SOX2, and E-cadherin, a signature of the epiblast, whereas expression of GATA4 and SOX17 was also activated moderately in dispersed cells, suggesting priming of these cells to endodermal development. Embedding the aggregates in Matrigel for further 3 days elicited migration of the cells into the lumen of laminin-rich matrices covering the aggregates, in which FOXA2 and SOX17 were expressed at a high level with the concomitant loss of E-cadherin, indicating the migratory phase of endodermal precursors. Prolonged culturing of the aggregates generated three segregating cell populations found in post-gastrulation stage embryos: (1) definitive endoderm co-expressing high SOX17, GATA4, and E-cadherin, (2) mesodermal cells expressing a low level of GATA4 and lacking E-cadherin, and (3) primed epiblast cells expressing POU5F1, SOX2 without E-cadherin. Thus, aggregation of EpiSCs followed by embedding of aggregates in the laminin-rich matrix models the gastrulation-dependent endoderm precursor development.

Chiral Sensing Using Circularly Polarized Luminescence of Bis(phenanthroline dicarboxylic acid) EuIII Complex Induced by Allosteric-type Interaction with Amino Acid Molecules.

[Eu(pda)2 ]- and [Eu(bda)2 ]- (pda=1,10-phenanthroline-2, 9-dicarboxylic acid; bda=2,2' bipyridine 5,5'-dicarboxylic acid) have an achiral D2d structure in crystals. These complexes exhibit circularly polarized luminescence (CPL) in water containing chiral amino acids. In this work, induced CPL of [Eu(pda)2 ]- and [Eu(bda)2 ]- in water solutions containing a mixture of d- and l- amino acids were examined. Plots of glum values of the induced CPL as a function of mol-fraction of l- and d- arginine reveal that [Eu(pda)2 ]- favors homo-association ([Eu(pda)2 ]- -(l-arginine)2 or [Eu(pda)2 ]- -(d-arginine)2 ) over hetero-association {[Eu(pda)2 ]- -(l-arginine)⋅(d-arginine)}. This suggests that association of an arginine molecule induces a structural change in [Eu(pda)2 ]- to promote chiral selective association to another arginine, i.e., homo-allosteric association. On the other hand, the system of [Eu(pda)2 ]- with histidine favors hetero-allosteric association over homo-association. No allosteric effect is recognized in CPL from [Eu(bda)2 ]- .

Perinatal risk in singleton pregnancies after in vitro fertilization.

OBJECTIVE: To assess perinatal risks to singleton births after in vitro fertilization (IVF) versus spontaneous conception. DESIGN: Cross-sectional. SETTING: A 2006 registry database of the Japan Society of Obstetrics and Gynaecology (JSOG) capturing 5.8% of total births. PATIENT(S): 53,939 singleton births from spontaneous conceptions and 1,408 singletons after IVF. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Perinatal death, low-birth weight (LBW), small for gestational age (SGA), congenital malformation, and sex ratio assessment based on singleton birth cases versus singleton live-born cases. RESULT(S): In this study, IVF may include intracytoplasmic sperm injection (ICSI), gamete intrafallopian transfer, or IVF followed by zygote intrafallopian transfer. In crude and adjusted analysis, perinatal death, SGA, congenital malformation, and sex ratio were not statistically significantly associated with IVF. The LBW rates were statistically significantly higher in IVF pregnancies, but the association statistically significantly decreased after adjusting for confounding factors. Placental previa, a maternal outcome, was found to be statistically significantly higher in IVF pregnancies. CONCLUSION(S): No evidence was implicated IVF procedures as dramatically increasing the adjusted risk of perinatal death, LBW, SGA, congenital malformation, or sex ratio when compared with spontaneous conceptions. However, because of sample size limitations, the study cannot exclude small to moderate increases in perinatal deaths or congenital malformations.