Because cannabis use has an age of onset SAMHSA that

Because cannabis use has an age of onset (SAMHSA, 2014) that overlaps with continued PFC gyrification development (Su et al., 2013; Klein et al., 2014), examining the impact of cannabis use on gyrification remains an important area to investigate. The current study examined whether cannabis use status predicted PFC or parietal gyrification in a sample of adolescents and emerging adults. Surface morphology may be related to cortical thickness and volume (Alemán-Gómez et al., 2013). Given that both reductions in cortical thickness and volume (Lopez-Larson et al., 2011; Price et al., 2015) and reductions in PFC sulcal concavity (Mata et al., 2010) were previously found in young cannabis users, we predicted that cannabis users would demonstrate reduced gyrification and SA in PFC and parietal regions. Reduced SA and gyrification may be most pronounced in both inferior frontal and parietal regions that show reductions in volume (Churchwell et al., 2010; Price et al., 2015) Within regions that differed between cannabis users and controls, follow-up analyses examined brain–behavior relationships in both groups.

Materials and methods

Results

Discussion
Decreased gyrification in right medial, SAG medial, and frontal pole regions, were associated with poorer performance on complex attention in cannabis users, suggesting that reduced gyrification confers a functional deficit. This is consistent with previous studies suggesting increased gyrification is associated with better cognitive functioning (Wallace et al., 2013) and may reflect improved cognitive control (Luders et al., 2012).
Present findings are consistent with prior research demonstrating unique PFC surface morphology characteristics in cannabis using youth (Mata et al., 2010). Specifically, Mata et al. (2010) found reduced sulcal concavity in the PFC of cannabis users in comparison to non-users and failed to find global hemispheric differences in SA. In the current study we found significantly reduced LGI in medial, ventral medial, and frontal poles in cannabis users compared to controls. We found no SAG significant differences in inferior parietal LGI and marginal differences in SA, while in an overlapping sample we previously reported subtle volume abnormalities in this region (Price et al., 2015). Though we did not examine the relationship between either LGI or SA and other cortical measures in this study, surface area, gyrification, and cortical thickness appear have distinct patterns in neurodevelopment from ages 6 to 22 (Raznahan et al., 2011). We also found unique patterns in cannabis effects between two cortical morphometry measures; after controlling for covariates including age and gender, results from the current study suggest that frequent cannabis use may influence LGI in a more diffuse PFC distribution compared to SA since we found only marginal reductions of SA in two PFC regions (left: ventral lateral and ventral medial PFC) among cannabis users compared to controls. Therefore, while gyrification may be partially related to gray matter volume and SA, it likely reflects a novel measure of brain maturation (Klein et al., 2014). Though Mata et al. (2010) did not find global hemispheric group differences in SA, perhaps the influence of frequent cannabis use on SA is restricted to regions with later SA development. Changes in global SA during late adolescents may be primarily driven by reduced global gyrification index (Raznahan et al., 2011) and may differ from influences driving cortical thickness maturation (Wierenga et al., 2014). Future studies may want to examine how cannabis use impacts neurodevelopment utilizing multiple measures of cortical morphometry (LGI, cortical thickness, volume, and SA).
Frequent cannabis-using youth report using cannabis to cope with stressors or relax (Boys et al., 2001; Mitchell et al., 2007; Bonn-Miller et al., 2007; Johnson et al., 2010; Benschop et al., 2015), although continued use may negatively impact regions underlying healthy affective processing (Etkin et al., 2011). For example, the medial portions of the PFC are implicated in self-referential thought, regulation of stress response, autonomic regulation, emotional processing, and social cognition (Urry et al., 2009; Somerville et al., 2013; Bado et al., 2014; for reviews see Uddin et al., 2007; Hänsel and von Känel, 2008). Ventral medial portions of the PFC play a role in regulating amygdala activity, contextual decision-making, fear response and extinction, anticipatory responses, and social processing (Aoki et al., 2014; Lonsdorf et al., 2014; Rudorf and Hare, 2014; Spoormaker et al., 2014; Motzkin et al., 2014, 2015). Animal studies suggest that the inferior frontal regions also play a vital role in insight or one\’s ability to imagine consequences of behavior in new situations (Lucantonio et al., 2012). The frontal pole underlies detecting contextual change, and reward-related decision-making (Pollmann and Manginelli, 2009; Kovach et al., 2012). Therefore, additional studies examining functional consequences of cannabis use in youth may focus on affective processing, reward processing, and mood symptomatology. In addition, given the potential impact of endocannabinoid signaling on PFC activation (Filbey et al., 2010), future studies may want to examine whether genotypes related to endocannabinoid signaling interact with cannabis exposure to predict frontolimbic structural integrity in youth.

Loss of the ApcWT allele

Loss of the ApcWT allele in intestinal SW033291 in organoid culture induces Wnt target genes and high self-renewal potential in these cells upon which they give rise to neoplastic growth. This manifests in cyst-like organoids with aberrant tissue architecture, cell differentiation and proliferation. In vivo loss of ApcWT in ISC leads to immediate induction of Wnt signaling and rapid expansion of ISCs resulting in aberrant tissue architecture and secretory cell differentiation typical of early intestinal adenomas in mice (Barker et al., 2009; Sansom et al., 2004). However, these studies used mice with an induced simultaneous loss of both Apc alleles, thus circumventing events that influence loss of Apc function first hand. Therefore, the spontaneous formation of cyst-like organoids genuinely reflects the initiating step in intestinal adenoma formation and represents a unique window to investigate cellular and molecular determinants of this first step in CRC. Our data suggest that the cyst-like ApcMin/Min organoids arise from an expansion of cells positive for the ISC marker Lgr5, but negative for the ISC marker Olfm4, a detail we find reflected in ApcMin/Min adenomas. This is in contrast to Olfm4-positive adenomas formed through Apc loss in Lgr5-positive cells (Myant et al., 2013; Schepers et al., 2012), suggesting that spontaneous adenomas in Min mice do arise through a mechanism differing from induced ApcWT-loss in ISCs.
A benefit of the organoid in vitro system is that it leaves out interactions of epithelial cells with stromal cells (Akcora et al., 2013; Barker et al., 2007), which can distort the detection of epithelial cell-specific signaling pathway interactions in vivo. Using this approach we wanted to know how the Notch and Myb pathways interact with Wnt signaling during the transition of ISC to tumor-initiating cells. We confirm findings of previous studies that loss-of-function of Notch (Pellegrinet et al., 2011; VanDussen et al., 2012) and Myb (Cheasley et al., 2011) strongly disturbs ISC self-renewal in vivo and in vitro. Surprisingly, we find that gain-of-function in these pathways does not increase self-renewal, suggesting that either both pathways alone are not sufficient to drive self-renewal, or their maximal capacity to do so, is limited and cannot be further increased in organoids.
Importantly we found that Notch activation increases the number of isolated Min crypts that process to cyst-like organoids, while Notch activation or inhibition does not affect growth or self-renewal once the cyst-like growth has been initiated. This is a striking parallel to the situation in vivo, where Notch activation increases the number of Wnt driven adenomas, but does not affect colon adenoma growth thereafter (Fre et al., 2009; Peignon et al., 2011). Intriguingly, we did not find any significant differences in SI adenoma formation across the genotypes on an Apcmin/+ background and we think this is because the activation of the Wnt pathway in the SI swamps out the effects of the other pathways. However, Notch activation and MybER had a significant effect in the colon in terms of adenoma formation and as colon cancer is approximately 10 times more common in humans we suggest that this was a more relevant matter to pursue. One explanation for this phenomenon is that Notch activation increases the number of tumor-initiating cells upon ApcWT-loss in the colon. This arguably can happen on three functional levels where we know aberrant Wnt activity affects initiation of intestinal adenomas: either by symmetric division of ISCs (Bellis et al., 2012), by the induction of a SC program in non-ISCs (Schwitalla et al., 2013) or by influencing the number of cells with ApcWT-loss able to clonally expand (Vermeulen et al., 2013). Notch activation leads to increased expression of the ISC markers Lgr5 and Olfm4, while the Lgr5-positive/Olfm4-negative phenotype of ApcMin/Min cyst-like organoids remains the same when Notch is activated and this is replicated in adenomas. This indicates that Notch-activation can affect the ISC phenotype, but does not affect the phenotype of tumor-initiating cells. Apparently the mechanism of how Notch influences adenoma initiation is complex and further studies will be necessary to elucidate it.

In the current study we compared

In the current study, we compared two different genetic backgrounds on the induction and maintenance of multipotency by generating clonal iNSC lines from two distinct mouse strains (C57BL/6 vs. C3H). Clonal iNSC lines from distinct genetic backgrounds exhibited the distinct levels of conversion efficiency, endogenous NSC markers, transgene expression, and in vitro differentiation although iNSC lines from both strains shared typical features of NSCs such as morphology, gene expression pattern, and epigenetic status, indicating that the genetic backgrounds influence the cell fate transition into an iNSC state. In addition, the comparative analysis of clonal iNSC lines could serve as a platform for screening the most suitable and functional iNSC line for clinical translation of direct conversion technology.

Materials and methods

Results

Discussion
To understand the mechanism underlying the induction of pluripotency, a number of inhibiting and boosting factors have been identified throughout screening multiple epigenetic and genetic factors potentially influencing iPSC generation. However, the factors that positively or negatively affect the induction of multipotency or unipotency via the direct conversion process, remain elusive. In this study, we tried to elucidate the effect of the genetic background on the induction and maintenance of iNSCs.
Although we were able to generate stably expandable iNSC lines from both mouse strains tested (C57BL/6 and C3H), these iNSC lines exhibited substantial differences in both the induction and maintenance phases of multipotency. First, the conversion efficiency as assessed by the number of SSEA1-positive population was significantly different between the two mouse strains. Furthermore, iNSCs from the C57BL/6 strain showed relatively poor induction of an iNSC state, as evidenced by impaired activation of endogenous Sox2 and incomplete silencing of transgenic Sox2. Thus, these data suggest that the genetic background influences the induction of multipotency in a neuronal lineage. Second, the survival rates of sorted SSEA1-positive single Atractyloside Dipotassium Salt was also significantly different between iNSC clones from the two strains, indicating that the maintenance of iNSC identity after successful conversion into the SSEA1-positive iNSC state is also influenced by the genetic backgrounds. Moreover, all clonal iNSC lines derived from C57BL/6 strain displayed the limited in vitro differentiation potential into neurons, astrocytes, and oligodendrocytes, indicating that the functionality of the iNSCs was also impaired in C57BL/6 iNSCs.
We observed the abnormally increased proliferation of C57BL/6 iNSC clones compared to C3H iNSCs and cNSCs (data not shown). In the previous study (Graham et al., 2003), it was demonstrated that the ectopic expression of Sox2 could induce increased proliferation and also block the proper differentiation of NSCs into their daughter cell types. Thus, the increased proliferation rate and impaired differentiation ability of C57BL/6 iNSCs can be explained by the residual expression of exogenous Sox2 in C57BL/6 iNSCs (Fig. 2A, Fig. 3C and Fig. S3). It would be interesting to further decipher the mechanism governing the strain-dependent regulation of both the endogenous NSC program and transgenes, which might be tightly linked to the functionality of iNSCs. Finally, it would be also interesting to examine whether the effect of the genetic background on the induction and maintenance of other cell types (e.g. induced hepatocytes and induced neurons) directly converted from somatic cells by defined transcription factors.
Recent studies have described that the distinct combinations of transcription factors can induce direct conversion of various human cells into an iNSC-like state (Lu et al., 2013; Ring et al., 2012; Wang et al., 2013; Yu et al., 2015; Zhu et al., 2014). Although the data provided in these studies support the cellular identity of human iNSCs, their molecular and functional features were not precisely characterized at the clonal level. Therefore, comparing the induction and maintenance of the NSC fate on human somatic cells at the clonal level might allow us to elucidate the underlying mechanism of direct conversion into NSCs.

Unlike the LMW FGF isoform which is well

Unlike the LMW FGF2 isoform, which is well-studied, the HMW isoforms of FGF2 are not nearly as well-understood, but recent reports have investigated the effects of HMW FGF2 signaling in various contexts. Localization of FGF2 isoforms can vary depending on cellular conditions and stimuli (Arese et al., 1999). HMW isoforms have been described in some occasions to be present in the extracellular space, released through a process known as vesicle shedding (Greber et al., 2007; Taverna et al., 2003). The vesicles have the potential to stimulate plasminogen activator production and influence chemotaxis (Taverna et al., 2003), suggesting a role for HMW FGF2 in this process, and previous studies have shown that the 24kDa FGF2 isoform administered exogenously to endothelial hydralazine hydrochloride induces cell proliferation with a similar dose response to the 18kDa isoform (Taverna et al., 2003), suggesting a potential role for extracellular, canonical FGFR signaling by HMW FGF2 isoforms. In HEK293 cells, overexpression of HMW FGF2 led to chromatin compaction and apoptosis in a manner dependent on its localization to the nucleus, further supporting the important role of subcellular localization in the biology of HMW FGF2 (Ma et al., 2007). Mice specifically overexpressing HMW FGF2 isoforms quickly develop osteoarthropathy, associated with dysregulated expression of inflammatory proteins and cytokines (Meo Burt et al., 2016), and HMW FGF2 has also recently been implicated in cardiac fibrosis and cardiomyocyte hypertrophy in rats (Jiang et al., 2007; Santiago et al., 2014). Previously, it had been demonstrated that, in neonatal rat cardiac non-myocytes, hypertrophic stimuli result in upregulation and subsequent secretion of HMW FGF2 to the extracellular space (Santiago et al., 2011). Taken together, these reports suggest non-redundant functions of HMW FGF2 compared to LMW FGF2, as well as the potential of HMW FGF2 to be present and active not only intracellularly, but also in the extracellular milieu in certain scenarios.
Human dermal fibroblasts express multiple isoforms of FGF2 as well as select FGF receptors (FGFRs) (Dailey et al., 2005; Dvorak et al., 2005; Eiselleova et al., 2009; Grose and Dickson, 2005; Quarto and Amalric, 1994; Root and Shipley, 2000). The LMW (18kDa) isoform of FGF2 has been studied most robustly and is reported to act in autocrine, intracrine, and paracrine manners, through interactions with its cell-surface receptors, the FGFRs (Ornitz et al., 1996; Zhang et al., 2006). Four distinct genes that encode for FGF receptors have been identified, FGFR1, FGFR2, FGFR3, and FGFR4 (Arese et al., 1999; Arnaud et al., 1999; Dvorak et al., 2005; Eiselleova et al., 2009; Florkiewicz and Sommer, 1989; Sperger et al., 2003; Taverna et al., 2003). Furthermore, alternative mRNA splicing produces several receptor variants, which exhibit varied binding kinetics and affinities for different FGF ligands (Champion-Arnaud et al., 1991; Miki et al., 1992; Root and Shipley, 2000). The variety of FGF ligands and FGFR variants provides a high level of diversity in ligand-binding specificity and biological function, depending on which FGFRs are expressed by various cell types and which FGFs are present in the surrounding milieu.
Binding of the FGF2 ligand to its receptor triggers receptor dimerization, phosphorylation of its kinase domain, and signal transduction via activation of several intracellular pathways that have been implicated in multiple aspects of vertebrate and invertebrate embryonic development, tumor growth, angiogenesis, wound healing, and physiology (Ornitz and Marie, 2002; Powers et al., 2000; Spivak-Kroizman et al., 1994). Dysregulated expression of FGFs has also been implicated in cancer development and progression (Ezzat and Asa, 2005; Givol and Yayon, 1992; Krejci et al., 2012; Presta et al., 2005; Zubilewicz et al., 2001). Investigation of the ability of each FGF isoform to bind to different FGF receptors and activate downstream signaling pathways, and identification of FGF-FGFR pair specificities, is critical for understanding the biological mechanisms involved in normal development and pathogenesis. Previous studies have identified the 18kDa FGF2 as an important factor for the maintenance of pluripotency in human stem cells (Eiselleova et al., 2009; Zoumaro-Djayoon et al., 2011), and our lab has demonstrated that the 18kDa FGF2 isoform, in combination with sub-atmospheric oxygen, induces expression of stem cell specific genes and proteins in human dermal fibroblasts cultured in vitro (Page et al., 2009).

gdc-0980 In a recent study Scheffer et al the

In a recent study (Scheffer et al., 2015), the expression of Lmod3 during natural mouse development was found to be restricted to cochlear HCs and that of Dnah5 to vestibular HCs, while double-positive cells were not produced. In the present study, immunocytochemical analyses showed that the cochlear HC marker Lmod3 and vestibular HC marker Dnah5 were expressed in 79.8±2.7% and 51.3±6.3%, respectively, of myosin6-immunopositive cells (Fig. 3D). These results suggested that some of the HC-like cells had co-expression of Lmod3 and Dnah5, though we unfortunately could not confirm their simultaneous expression because an appropriate set of gdc-0980 to recognize them was not available. Restricted expression of Lmod3 and Dnah5, as observed in naturally developed HCs (Scheffer et al., 2015), was not recapitulated in the present combined method. Continuous overexpression of Math1 during the culture period explains the dysregulated expression of Lmod3 and Dnah5. Math1 expression in the cochlea begins on embryonic day (ED) 13.5 and continues until postnatal day 3–7, or possibly even into adulthood (Lumpkin et al., 2003; Lanford et al., 2000; Scheffer et al., 2007). On the other hand, Math1 expression in vestibular cells begins on ED12.5, then spreads throughout sensory epithelium by E14.5 and is down-regulated during the first postnatal week (Shailam et al., 1999; Bermingham et al., 1999). To recapitulate reproduce the expression pattern of the Math1 gene that occurs during natural development, precise regulation of its expression may be required. Recently, use of small molecular weight compounds such as DAPT has been reported to increase endogenous Math1 gene expression (Ren et al., 2016). Therefore, instead of direct gene induction by gene transfer, chemicals known to be Math1 inducers may function well with gene-engineered ES cells and simplify the present method.

Conclusion
In summary, we investigated the efficacy of a combination of Math1-ES cells and use of ST2-CM, termed the HIST2 method, for inducing HC-like cells. Our results showed that Math1 expression increased the production of both cochlear- and vestibular-HC-like cells.
The following are the supplementary data related to this article.

Authors\’ contributions

Conflicts of interest

Acknowledgments
We thank Dr. H. Niwa (Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan) for the ES cell lines and S. Shimada for the technical assistance. This research was supported by the Osaka Medical Research Foundation for Intractable Diseases and Grant-in-Aid for Scientific Research (B) (to Y.O. and M.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI) (16H05482).

Introduction
Over the past few years, several studies using hPS cells focused on the use of transgene-free strategies to recapitulate embryonic skeletal myogenesis in vitro (Barberi et al., 2007; Chal et al., 2016; Hwang et al., 2013; Shelton et al., 2014; Xi et al., 2017; Zheng et al., 2006). However, most of the current transgene-free myogenic differentiation protocols have limitations for potential clinical translation. The primary problems are the heterogeneity of cell preparations and the lack of evidence for the in vivo regenerative potential of generated myogenic cells (Kim et al., 2017). To date, the most homogeneous approaches for the generation of a myogenic-restricted population from PS cells relies on the introduction of exogenous DNA for the myogenic regulatory factor MYOD (Albini et al., 2013; Goudenege et al., 2012; Tedesco et al., 2012; Young et al., 2016) or PAX7 (Darabi et al., 2012; Kim et al., 2017), a paired box transcription factor essential for the commitment and maintenance of muscle stem cells (Gros et al., 2005; Lagha et al., 2008; Seale et al., 2000).
Few of these studies have documented transplantation of PS-cell derived progenitors, but of those that have, most have shown minimal engraftment (Barberi et al., 2007; Hwang et al., 2013; Kim et al., 2017; Zheng et al., 2006). In contrast to these studies, the approach of temporally overexpressing PAX7 enables the generation of large numbers of proliferating PAX7+ myogenic progenitors and when these are transplanted, they contribute to significant skeletal muscle regeneration (Darabi et al., 2012; Kim et al., 2017; Magli et al., 2017). To date, the PAX7 approach has used lentiviral (LV) delivery of PAX7, which introduces the possibility of insertional mutagenesis.

br Experimental Procedures br Author Contributions br Acknowledgments

Experimental Procedures

Author Contributions

Acknowledgments

Introduction
The cardiac conduction system (CCS) is composed of the impulse-generating sino-atrial (SA) and atrio-ventricular (AV) nodes and the impulse-propagating His-Purkinje system, which play critical roles initiating and regulating excitation and contraction of the cardiac chambers. Dysfunction of the CCS caused by disease, congenital malformations, aging, or heritable gene defects results in cardiac arrhythmias, a major cause of morbidity and mortality worldwide (Spooner et al., 2001). Current treatments of cardiac arrhythmias include pharmacological therapy and implantable rhythm-management devices, such as pacemakers and defibrillators. However, these treatments can have low efficacy and also cause additional problems, such as pro-arrhythmia side effects, infection, and device malfunction (Holzmeister and Leclercq, 2011; Woods and Olgin, 2014). Devices placed into pediatric patients must be replaced as heart development proceeds throughout life. Thus, there is a clear and compelling need to develop strategies to treat patients with CCS defects.
Genetic and cell-based therapeutic approaches have focused on generation of biological pacemakers (Cho and Marbán, 2010; Rosen et al., 2004). Embryonic stem cells (ESCs) are a feasible starting material, as they are pluripotent and can be differentiated to any cell lineage. Several ESC reporter lines have been engineered and used to isolate CCS cells, including (1) HCN4p-EGFP+ cells that express transcripts for CCS markers Hcn4, Cav3.2, and Cx40 and display a hyperpolarization-activated depolarizing current (termed the funny current; Morikawa et al., 2010); (2) an ESC reporter line containing a Mink:lacz transgene and a reporter regulated by a chicken Gata6 (cGata6) enhancer; the double-positive cells display pacemaker-like morphology and show pacemaking chk1 inhibitor waveform as well as a funny current (White and Claycomb, 2005); (3) a Shox2 promoter with a Cx30.2 enhancer to generate ESC-derived SHOX2+ and Cx30.2+ cardiomyocytes that express additional CCS markers (Hcn4, Cx45, Cx30.2, Tbx2, and Tbx3) and exhibit pacemaker-nodal phenotype (Hashem and Claycomb, 2013); (4) ESC-derived Nkx2.5:GFP+ cells expressing Hcn1 and Hcn4 that display the funny current (Yano et al., 2008); and (5) an ESC reporter line derived from Contactin2:EGFP BAC transgenic mice (Cntn2:egfp) was recently used to isolate rare cells that display transcriptional signatures and functional properties comparable to endogenous cardiac Purkinje cells (PCs) (Maass et al., 2015). Finally, CD166 has been identified as a CCS cell-surface marker (Scavone et al., 2013), although it is not specific to CCS constituents.
ESCs can also be directed into CCS-like cells using chemicals and small-molecule compounds. For example, vitamin B12 promoted the expression of CCS markers Cx40 and Hcn4 at an early stage of cardiomyocyte differentiation (Saito et al., 2009). Treating ESCs with Ca2+-activated potassium channel (SKCa) activator, 1-ethyl-2-benzimidazolinone (EBIO), or suramin promoted a nodal-like cell phenotype (Kleger et al., 2010; Wiese et al., 2011). Hence, cell-permeable small molecules that modulate functions of specific pathways provide a convenient and efficient approach to control stem/progenitor cell fate. Importantly, these small molecules provide new tools to dissect molecular mechanisms that control embryonic development, therefore facilitating a better understanding for functions of relevant signaling pathways. However, overall efficiency of generating CCS cells using any of the current protocols is poor (typically below 1% of the culture). Thus, developing an efficient strategy to derive CCS cells will not only facilitate developing disease models for mechanistic studies and drug discovery but also provide new cellular materials for regenerative therapy.
Here, we describe a high-throughput screen of ∼5,000 compounds using an ESC line derived from the CCS:lacz reporter mouse, containing a transgene that fortuitously marks cells of the CCS lineage (Rentschler et al., 2001). We discovered that the small molecule sodium nitroprusside (SN) efficiently enhances the generation of CCS cells from ESCs. The screen was validated using an additional reporter line, with GFP expression driven by a Contactin2 (Cntn2) transgene (Maass et al., 2015). The derived GFP+ cells induced by SN display an expression profile, molecular markers, and functional properties that are comparable to endogenous PC of the ventricular CCS. Importantly, we found that SN promotes the generation of PC-like cells through activating cyclic AMP (cAMP) signaling in embryonic cardiac cells, providing new insight into the ontogeny of CCS lineages.

tropisetron There is now compelling evidence indicating that

There is now compelling evidence indicating that both the EMP and definitive programs derive from progenitors known as hemogenic endothelial tropisetron (HECs) that display endothelial markers and are associated with the arterial vasculature (Bertrand et al., 2005; Boisset et al., 2010; Clarke et al., 2013; Kissa and Herbomel, 2010). Live imaging and histological analyses have demonstrated that hematopoietic progenitors develop from the HECs through a process known as the endothelial to hematopoietic transition (EHT) (Boisset et al., 2010; Kissa and Herbomel, 2010). Currently, there are no markers to distinguish EMP-HECs from definitive HECs. As the EMP program develops earlier than the definitive program, the EMP-derived lineages can predominate the PSC differentiation cultures and are often misinterpreted as representing definitive hematopoiesis. Developing strategies to distinguish definitive- from EMP-derived hematopoiesis in the PSC differentiation cultures is essential for identifying the signaling pathways that regulate the development of definitive HECs and their specification to HSCs.
We have previously shown that SOX17 is expressed in definitive HECs and that it is required for the establishment of the definitive, but not the primitive, hematopoietic program (Clarke et al., 2013). Our analysis of differentiated hematopoietic progeny from HECs generated from SOX17-null mouse embryonic stem cells (mESCs) revealed the presence of primitive erythroid progenitors, myeloid progenitors, and erythroid progenitors that gave rise to large colonies distinct from the primitive erythroid colonies. The presence of this population of erythroid progenitors suggests that the EMP program may also be Sox17 independent. Our goals in the present study were to define the role of Sox17 in EMP hematopoiesis, understand how Sox17 regulates the generation of definitive hemogenic endothelium, and identify additional markers to distinguish these programs. To achieve these goals, we deciphered the proteome of AGM-derived HECs and of HECs generated from Sox17-wild-type and Sox17-knockout mESCs using label-free quantitative proteomics. The findings from these studies demonstrate the overall utility of analyzing the proteome of small numbers of cells and show that expression of the transcription factor Stat1 and dependency on Sox17 distinguish the definitive erythroid lineage from the primitive and EMP-derived erythroid lineages.

Results

Discussion
Label-free proteomics represents an emerging technology for identifying and quantifying patterns of protein expression. While differences in patterns of protein expression have previously been shown to correlate with differentiated cell types, it has been much more challenging to develop technologies that can detect regulatory proteins such as transcription factors that specify specific cellular fates. Emerging embryonic HSCs and their progeny have been comprehensively examined via global transcriptional profiling, which revealed key insights into hematopoietic specification and leukemogenesis (Boisset et al., 2010; McKinney-Freeman et al., 2012). The direct precursor to the HSC, the HEC, has not been extensively examined, as only an abbreviated transcriptional profile of HECs within the murine embryo was recently elucidated using a Fluidigm-based approach (Swiers et al., 2013). Here, we have utilized label-free proteomics to decipher key regulators of embryonic hematopoiesis both in vivo and during the directed differentiation of ESCs in vitro. This approach to cellular profiling has the distinct advantage of identifying only translated functional proteins present in the cell, thus eliminating the unknown effects of post-transcriptional modification that may affect mRNA translation.
Our first analysis focused on the proteomic landscape of E10.5 and E11.5 VEC+ populations that contain ECs, HECs, and pre-HSCs (Rybtsov et al., 2011). With this approach, we were able to accurately identify and quantify known regulators of the early hematopoietic programs such as SCL/TAL1 and ALDH1A2, as well as a small cohort of proteins that exhibited dramatic expression changes between E10.5 and E11.5, providing a focused and manageable list of candidates to interrogate as putative regulators of the generation of type I pre-HSCs. To date, the generation of PSC-derived HSCs has been difficult, likely due to the undefined nature of PSC differentiation culture conditions and the lack of accurate comparison of the in vitro populations to potential equivalent in vivo embryonic stages. Comparison of D7 mESC-derived HEC populations to the E10.5 and E11.5 HEC populations revealed that our D7 HEC population represents a developmental intermediate. Therefore, interrogation of this dataset will help identify the absence of key regulatory proteins, thus providing a potential explanation for the inability of these cells to faithfully generate HSCs in vitro.

Despite the dramatic changes in bone morphology in

Despite the dramatic changes in bone morphology in OsxCre;iDTR mutants, bone marrow cellularity was unaffected compared with their control littermates (Figure 3A). Red blood cell and platelet numbers were unchanged in the blood of the OsxCre;iDTR mutant, but there was a 50% decrease in the white blood cell count due to severe lymphopenia (Figure 3B). The mutant bone marrow had fewer mature B220+IgM+ B cells, increased monocytes and granulocytes (Figure 3A), and a corresponding increase in granulocyte and macrophage progenitors (GMPs) (Figure 3C). Given the importance of the spleen for B lymphopoiesis and extramedullary hematopoiesis, we analyzed the spleens and noted no difference in weight, but similarly increased Mac1+Gr1+ cells in the OsxCre;iDTR mutants (Figure 3D). In summary, Osx cell deletion caused a loss of mature Tasquinimod and an increase in myeloid cells in both the bone marrow and spleen of mutant animals.
We assessed whether the GMP increase in the OsxCre;iDTR mutants was secondary to increased proliferation or decreased death of GMPs. There was change in neither cell-cycle status (Figure S3A) nor apoptosis (Figure S3B). The accumulation of GMP and Mac1+Gr1+ cells could either be an osteoprogenitor-mediated hematopoietic effect or a consequence of inflammatory phagocytic-type immune response triggered by the death of osteolineage cells. To test this hypothesis, we co-injected indomethacin, a non-steroidal anti-inflammatory drug, together with DT in OsxCre;DTR mutant mice for 1 week. Bone marrow analysis revealed normalization of the GMP, Mac1+, and Mac1+Gr1+ cell populations in indomethacin-treated animals (Figure S3C). Therefore, the increase in Mac1+Gr1+ myeloid cells could be due to inflammatory response to osteolineage cell death, although we cannot exclude an osteolineage cell-mediated myeloid effect.
We asked whether HSC function was affected by the short-term deletion of Osx cells. We observed no differences between the OsxCre;iDTR mutants and controls in long-term HSC (LINloC-KIT+SCA-1+CD48−CD150+) number (Figure S4A), proliferation (Figure S4B), or apoptosis (Figure S4C). There was an increase in LINloC-KIT+SCA-1− progenitor cell number and cell cycle (Figures S4A and S4B). However, no defect in HSC function was detected when bone marrow cells from were transplanted into primary and secondary recipients in a 1:1 ratio with congenic SJL competitor cells (Figure S4D). These data suggest that short-term deletion of Osx cells did not affect HSC function.
Given the diminished production of mature B cells, we assessed CLPs and found no difference (Figure 3C). However, intermediate stages of B cell development were affected. Specifically, a decrease in both pre-B and mature B populations were noted while pro-B cells, especially during the later C′ pro-B and C″ pro-B stages, were significantly increased (Figure 4A). These data strongly suggest that B cell differentiation was impaired at the pro-B to pre-B juncture.
We next tested whether these changes affected the animal\’s immune response. Mice were challenged with NP-Ficoll to trigger a T cell-independent (Maizels et al., 1988) immune response and followed for 28 days. Data revealed that OsxCre;iDTR mutants could not sustain immunoglobulin G (IgG) and M (IgM) production over time compared with their control littermates (Figures 4B and 4C). These data indicate that the perturbation in B cell development was not simply immunophenotypic, but there was also a functional reduction in Ig production and immune response.
To determine whether the B cell differentiation defect was microenvironment-dependent, we transplanted 1 × 106 bone marrow cells from congenic SJL mice into lethally irradiated control or mutant OsxCre;iDTR recipients. A similar rate of reconstitution was seen in both control and mutants (Figure 5A). As wild-type SJL cells repopulated both control and mutant OsxCre;iDTR recipients over the next 12 weeks, we observed a gradual recapitulation of the OsxCre+/−;DTRFl/+ hematopoietic phenotype in the mutant recipients but not in the controls (Figures 5B–5F). These data strongly suggest that the incomplete B cell differentiation observed in OsxCre;iDTR was caused by an altered bone microenvironment.

br Summary A new niobium diffusion

Summary
A new niobium BTS supplier mechanism at the copper–niobium interfacial boundary characterized by the displacement of atoms along the mismatch dislocations has been established. Diffusion processes in niobium are activated by the introduction of a number of copper atoms in the rows of niobium atoms near the mismatch dislocations. The activation energy of the process within the error corresponds to that of the copper diffusion along the high-angle grain boundary of niobium. Up to the temperature of 1200K, the vacancy mechanism of diffusion at the Cu/Nb boundary does not play a significant role.

Acknowledgment
The work was financially supported by the Ministry of Education and Science of the Russian Federation under contract No. 3.1282.2014/K.

Introduction
One of the outstanding implications of introducing hydrogen into metal is the possibility of increasing of the vacancy concentration in this material by several orders of magnitude, the so-called superabundant vacancy (SAV) formation. The physical kernel of this effect is a trapping of hydrogen atoms into vacancies, leading to a decrease in the vacancy formation energy. In 1993 the SAV was first discovered experimentally by Fukai and co-workers in Pd [1] and Ni [2]. Since then the SAV has been observed in many metallic systems such as Cu [3], Ti [4], Pd and Pd alloys [5–8], Al [9], Mn [10], Fe [10,11], Mo [12], Cr [13], Co [10], Ni [14], Ni-Fe alloy [15], Nb [16–18].
In some cases, the concentration of vacancies can become as large as 10% and more [10,19,20]. One of the reasons for this behavior is the formation of different defects, e.g., voids [21] as in the case of Al [9], Ni [22], Fe [23,24], steels [25], and/or dislocation loops [26–28].
It is important to note, that H atoms may have a high number of configurations inside a vacancy, leading to a higher configuration entropy of a crystal with H-vacancy complexes compared with a crystal with single vacancies and interstitial H atoms. To investigate the effects of SAV formation on material from a theoretical point of view, one has to consider the positions of hydrogen atoms inside a vacancy and the maximum number of hydrogen atoms that can be trapped by vacancy. In the aluminum lattice, it was observed that a hydrogen atom BTS supplier diffuses towards the lattice vacancy to occupy it [29]. Instead of occupying the lattice site from which an aluminum atom was removed during vacancy formation, the hydrogen atom locates at some asymmetrical point which is away from the vacancy\’s center. Lu and Kaxiras [30] suggested that, in principle, up to 12 atoms can be accommodated in a single vacancy in the Al lattice. More recently, Gunaydin, et al. [31] reported that trapping of multiple H atoms in a single vacancy in Al is possible only for extreme H loading condition, i.e., for H concentration many orders of magnitude above the solubility limit given by the equilibrium with H2 gas at ambient conditions (10−9 at 300K and 1 atm). Instead, under H-loading conditions used in most diffusion experiments, empty vacancies would coexist with the vacancies filled by one or at most two H atoms. Reviewing the conflicting results, from Lu and Kaxiras [30] on one hand and from Gunaydin et al. [31] on the other hand, Lars Ismer, et al. studied the interactions between hydrogen impurities and vacancies in FCC Al lattice by first-principle (ab initio) methods but with a more critical estimation on the binding energy of the H-vacancy complex [32]. They found that a single vacancy in Al lattice can trap 10 hydrogen atoms, not 12 as reported in the work of Lu and Kaxiras [30]. The difference occurs because of the definition of the trapping energy of hydrogen impurities in vacancies of each author. However, we can see the common result in the two works that hydrogen atoms can interact with the vacancies forming H-vacancy complexes. Moreover, trapping of multiple H atoms in a single vacancy in Al lattice decreases the vacancy formation energy and this phenomenon could be related to the observed hydrogen-induced superabundant vacancy formation and vacancy clustering.

br Data and methods To assess these possibilities we analyze

Data and methods
To assess these possibilities we analyze data from the China Family Panel Studies (CFPS), and include information from interviews conducted in 2010 (Wave 1) and 2012 (Wave 2). The CFPS uses a nationally representative multi-stage probability sample, with counties (or equivalent) as the primary sampling units, villages (or equivalent) sampled within counties, and households sampled within villages. The 2010 baseline survey included 14,960 households, including 33,600 adult respondents aged 16 and older, and 8990 children aged under 16, with an individual response rate of roughly 84 percent (Xie, 2012).
Approximately 80.6 percent of those who took part in Wave 1 were successfully tracked and re-interviewed for Wave 2 (Xie & Hu, 2014). Roughly 20 percent of the full sample was thus lost to attrition. Unfortunately, in panel studies, this is not unusual. In our subsample of those aged 50 and older at Time 1 who have complete information on all variables included in the analysis, the attrition was slightly more substantial (73 percent of 2010 respondents were tracked and re-interviewed). Not surprisingly, it hcv protease inhibitor was also selective on certain of our key indicators, including older age and marital status, in particular having been widowed at Time 1. As a consequence, those who were interviewed in Wave 2 are likely healthier than those lost to attrition and our results should be read accordingly.

Results

Summary and conclusions
We contribute to the literature in a number of ways. We have three indicators of health and well-being that, combined, provide a compelling picture of the importance of having a partner as one enters older ages in the context of China. Although one might expect analyses of depression and life satisfaction to draw similar conclusions, our results suggest that these variables capture several distinct aspects of life for older adults in China. By and large, we find that those who were formally married at both points in time fared better than those who were not, and than those whose unions dissolved through divorce or widowhood during the interval. Thus both the marital resources argument and the crisis argument have received some support (Goldman, Korenman, & Weinstein, 1995; Williams & Umberson, 2004). We find very little evidence to suggest that the presence or absence of children effectively buffers the marital status effects, although we do observe some positive effects on health of intergenerational co-residence. In addition, we find some important differences according to gender, both in the physical and emotional health reports of men and women, and in what predicts better outcomes for each at older ages. We are able to control for Time 1 health indicators to reduce the possibility that selection effects regarding health are operating.
Several specific findings pertaining to marital status are worth reemphasizing. First, counter to expectations, we observed few significant differences between being in a cohabiting partnership and being in formal/legal one, except on the measure on life satisfaction. We did find that men who were in cohabiting unions at both time points were less satisfied with life than were their legally married counterparts, net of presence of children and other factors, but this is based on a very small number of cases and, as we have said, is a finding that must be read with caution. Being without a partner at all, either because one was never-married, or because one was widowed or divorced, was much more hcv protease inhibitor consequential for health and emotional well-being. For example, physical health was adversely affected by being never married (mainly for men) and by becoming widowed in the interval (mainly for women). Becoming widowed also resulted in worse depression and lower life satisfaction, mainly for women. These findings should be a matter of ongoing policy concern. Although based on very few cases, it also appears that becoming divorced in the interval may have especially detrimental effects on women (a result that should also be interpreted with caution). We noted earlier that divorce rates have risen in China in recent years, and the effects of divorce on health should be studied in future research with larger samples of divorced respondents.