Characterization of migratory primordial germ cells in the aortagonad-mesonephros of a 4.5-week-old human embryo : a toolbox to evaluate in vitro early gametogenesis

STUDY QUESTION: Which set of antibodies can be used to identify migratory and early post-migratory human primordial germ cells (hPGCs)? STUDY FINDING: We validated the specificity of 33 antibodies for 31 markers, including POU5F1, NANOG, PRDM1 and TFAP2C as specific markers of hPGCs at 4.5 weeks of development of Carnegie stage (CS12-13), whereas KIT and SOX17 also marked the intra-aortic hematopoietic stem cell cluster in the aorta-gonad-mesonephros (AGM). WHAT IS KNOWN ALREADY: The dynamics of gene expression during germ cell development in mice is well characterized and this knowledge has proved crucial to allow the development of protocols for the in vitro derivation of functional gametes. Although there is a great interest in generating human gametes in vitro, it is still unclear which markers are expressed during the early stages of hPGC development and many studies use markers described in mouse to benchmark differentiation of human PGC-like cells (hPGCLCs). Early post-implantation development differs significantly between mice and humans, and so some germ cells markers, including SOX2, SOX17, IFITM3 and ITGA6 may not identify mPGCs and hPGCs equally well. STUDY DESIGN, SIZE, DURATION: This immunofluorescence study investigated the expression of putative hPGC markers in the caudal part of a single human embryo at 4.5 weeks of development. PARTICIPANTS/ MATERIALS, SETTING, METHODS: We have investigated by immunofluorescence the expression of a set of 33 antibodies for 31 markers, including pluripotency, germ cell, adhesion, migration, surface, mesenchymal and epigenetic markers on paraffin sections of the caudal part, including the AGM region, of a single human embryo (CS12-13). The human material used was anonymously donated with informed consent from elective abortions without medical indication. MAIN RESULTS AND THE ROLE OF CHANCE: We observed germ cell specific expression of NANOG, TFAP2C and PRDM1 in POU5F1+ hPGCs in the AGM. The epigenetic markers H3K27me3 and 5mC were sufficient to distinguish hPGCs from the surrounding somatic cells. Some mPGC-markers were not detected in hPGCs, but marked other tissues; whereas other markers, such as ALPL, SOX17, KIT, TUBB3, ITGA6 marked both POU5F1+ hPGCs and other cells in the AGM. We used a combination of multiple markers, immunostaining different cellular compartments when feasible, to decrease the chance of misidentifying hPGCs. LARGE SCALE DATA: Non-applicable. LIMITATIONS REASONS FOR CAUTION: Material to study early human development is unique and very rare thus restricting the sample size. We have used a combination of antibodies limited by the number of paraffin sections available. WIDER IMPLICATIONS OF THE FINDINGS: Most of our knowledge on early gametogenesis has been obtained from model organisms such as mice and is extrapolated to humans. However, since there is a dedicated effort to produce human artificial gametes in vitro, it is of great importance to determine the expression and specificity of human-specific germ cell markers. We provide a systematic analysis of the expression of 31 different markers in paraffin sections of a CS12-13 embryo. Our results will help to set up a toolbox of markers to evaluate protocols to induce hPGCLCs in vitro

Snoring in Portuguese primary school children

OBJECTIVE: To determine the prevalence of snoring and its potential associations with sleep problems, such as daytime symptoms, medical conditions, school performance, and behavioral disturbances in Portuguese children attending primary school. METHODS: A previously validated questionnaire was sent to the parents of 1381 children attending primary schools in a parish of Coimbra, Portugal. To assess behavioral disturbances, the Portuguese version of Rutter's Children's Behavior Questionnaire for completion by teachers was used. RESULTS: Of the 988 questionnaires returned (71.5%), complete information concerning snoring was obtained for 976 children (496 girls and 480 boys; mean age: 8.1 +/- 1.5 years). Loud snoring during sleep was reported as frequent or constantly present (LSn) in 84 children (8.6%), as occasionally present in 299 children (30.6%), and as never present (NSn) by 593 children (60.8%). The LSn and NSn groups did not differ with respect to age, gender, sleep duration, time to fall asleep, frequency of night wakings, bedwetting, daytime tiredness, and school achievement. However, LSn was significantly associated with increased bedtime problems (fears and struggles), increased need for comforting activities to fall asleep, behaviors suggestive of parasomnias (sleep talking, teeth grinding, and night terrors), increased daytime sleepiness and irritability, and behavioral disturbances. Children in the LSn group were also more likely to report recurrent medical problems particularly those involving infections of the respiratory tract. CONCLUSIONS: Snoring is a common symptom in Portuguese children that is associated with behavioral daytime and sleep time disturbances. Children with loud snoring may benefit from early evaluation and intervention

Game theory of mind

This paper introduces a model of ‘theory of mind’, namely, how we represent the intentions and goals of others to optimise our mutual interactions. We draw on ideas from optimum control and game theory to provide a ‘game theory of mind’. First, we consider the representations of goals in terms of value functions that are prescribed by utility or rewards. Critically, the joint value functions and ensuing behaviour are optimised recursively, under the assumption that I represent your value function, your representation of mine, your representation of my representation of yours, and so on ad infinitum. However, if we assume that the degree of recursion is bounded, then players need to estimate the opponent's degree of recursion (i.e., sophistication) to respond optimally. This induces a problem of inferring the opponent's sophistication, given behavioural exchanges. We show it is possible to deduce whether players make inferences about each other and quantify their sophistication on the basis of choices in sequential games. This rests on comparing generative models of choices with, and without, inference. Model comparison is demonstrated using simulated and real data from a ‘stag-hunt’. Finally, we note that exactly the same sophisticated behaviour can be achieved by optimising the utility function itself (through prosocial utility), producing unsophisticated but apparently altruistic agents. This may be relevant ethologically in hierarchal game theory and coevolution

Evolution of Xylan Substitution Patterns in Gymnosperms and Angiosperms: Implications for Xylan Interaction with Cellulose.

The interaction between cellulose and xylan is important for the load-bearing secondary cell wall of flowering plants. Based on the precise, evenly spaced pattern of acetyl and glucuronosyl (MeGlcA) xylan substitutions in eudicots, we recently proposed that an unsubstituted face of xylan in a 2-fold helical screw can hydrogen bond to the hydrophilic surfaces of cellulose microfibrils. In gymnosperm cell walls, any role for xylan is unclear, and glucomannan is thought to be the important cellulose-binding polysaccharide. Here, we analyzed xylan from the secondary cell walls of the four gymnosperm lineages (Conifer, Gingko, Cycad, and Gnetophyta). Conifer, Gingko, and Cycad xylan lacks acetylation but is modified by arabinose and MeGlcA. Interestingly, the arabinosyl substitutions are located two xylosyl residues from MeGlcA, which is itself placed precisely on every sixth xylosyl residue. Notably, the Gnetophyta xylan is more akin to early-branching angiosperms and eudicot xylan, lacking arabinose but possessing acetylation on alternate xylosyl residues. All these precise substitution patterns are compatible with gymnosperm xylan binding to hydrophilic surfaces of cellulose. Molecular dynamics simulations support the stable binding of 2-fold screw conifer xylan to the hydrophilic face of cellulose microfibrils. Moreover, the binding of multiple xylan chains to adjacent planes of the cellulose fibril stabilizes the interaction further. Our results show that the type of xylan substitution varies, but an even pattern of xylan substitution is maintained among vascular plants. This suggests that 2-fold screw xylan binds hydrophilic faces of cellulose in eudicots, early-branching angiosperm, and gymnosperm cell walls.This work was supported by the Leverhulme Trust Centre for Natural Material Innovation (MBW, PD), The Low Carbon Energy University Alliance (AL), BBSRC Grant: BB/G016240/1 BBSRC Sustainable Bioenergy Centre cell wall sugars (TT, PD) and the Sao Paulo Research Foundation (RLS, CSP, MSS, TCFG) (Grants 2013/08293-7, 2014/10448-1 and 2015/25031-1)

The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana.

The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan-cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy.The work conducted by TT and NN was supported by a grant from the BBSRC: BB/G016240/1 BBSRC Sustainable Energy Centre Cell Wall Sugars Programme (BSBEC) to PD and DNB. The work of PD was supported by the European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013) under the grant agreement #251132. The NMR facility infrastructure was supported by the BBSRC and the Wellcome Trust. TCFG thanks CNPq (Brazil) for a graduate fellowship (grant # 140978/2009-7). MSS thanks CEPROBIO (grant # 490022/2009- 0) and FAPESP for funding (grant #2013/08293-7).This is the accepted version of the following article: "Busse-Wicher, M; Gomes, T.C.F; Tryfona, T; Nikolovski, N; Stott, K; Grantham, N.J; Bolam, D.N; Skaf, M.S; Dupree, P. (2014) "The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a two-fold helical screw in the secondary plant cell wall of Arabidopsis thaliana." The Plant Journal. Accepted article [electronic] 10.1111/tpj.12575", which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1111/tpj.12575/abstrac

A novel multivariate STeady-state index during general ANesthesia (STAN)

The assessment of the adequacy of general anesthesia for surgery, namely the nociception/anti-nociception balance, has received wide attention from the scientific community. Monitoring systems based on the frontal EEG/EMG, or autonomic state reactions (e.g. heart rate and blood pressure) have been developed aiming to objectively assess this balance. In this study a new multivariate indicator of patients' steady-state during anesthesia (STAN) is proposed, based on wavelet analysis of signals linked to noxious activation. A clinical protocol was designed to analyze precise noxious stimuli (laryngoscopy/intubation, tetanic, and incision), under three different analgesic doses; patients were randomized to receive either remifentanil 2.0, 3.0 or 4.0 ng/ml. ECG, PPG, BP, BIS, EMG and [Formula: see text] were continuously recorded. ECG, PPG and BP were processed to extract beat-to-beat information, and [Formula: see text] curve used to estimate the respiration rate. A combined steady-state index based on wavelet analysis of these variables, was applied and compared between the three study groups and stimuli (Wilcoxon signed ranks, Kruskal-Wallis and Mann-Whitney tests). Following institutional approval and signing the informed consent thirty four patients were enrolled in this study (3 excluded due to signal loss during data collection). The BIS index of the EEG, frontal EMG, heart rate, BP, and PPG wave amplitude changed in response to different noxious stimuli. Laryngoscopy/intubation was the stimulus with the more pronounced response [Formula: see text]. These variables were used in the construction of the combined index STAN; STAN responded adequately to noxious stimuli, with a more pronounced response to laryngoscopy/intubation (18.5-43.1 %, [Formula: see text]), and the attenuation provided by the analgesic, detecting steady-state periods in the different physiological signals analyzed (approximately 50 % of the total study time). A new multivariate approach for the assessment of the patient steady-state during general anesthesia was developed. The proposed wavelet based multivariate index responds adequately to different noxious stimuli, and attenuation provided by the analgesic in a dose-dependent manner for each stimulus analyzed in this study.The first author was supported by a scholarship from the Portuguese Foundation for Science and Technology (FCT SFRH/BD/35879/2007). The authors would also like to acknowledge the support of UISPA—System Integration and Process Automation Unit—Part of the LAETA (Associated Laboratory of Energy, Transports and Aeronautics) a I&D Unit of the Foundation for Science and Technology (FCT), Portugal. FCT support under project PEst-OE/EME/LA0022/2013.info:eu-repo/semantics/publishedVersio

Short-Term Analysis of Human Dental Pulps After Direct Capping with Portland Cement

This study evaluated the short-term response of human pulp tissue when directly capped with Portland cement. In this series of cases, twenty human third molars that were scheduled for extraction were used. After cavity preparation, pulp exposure was achieved and Portland cement pulp capping was performed. Teeth were extracted after 1, 7, 14 and 21 days following treatment and prepared for histological examination and bacterial detection. Each group had 5 teeth. The results were descriptively analysed. Dentin bridge formation was seen in two teeth with some distance from the material interface (14 and 21 days). Soft inflammatory responses were observed in most of the cases. Bacteria were not disclosed in any specimen. PC exhibited some features of biocompatibility and capability of inducing mineral pulp response in short-term evaluation. The results suggested that PC has a potential to be used as a less expensive pulp capping material in comparison to other pulp capping materials