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Environmental education is implemented in formal curricula in multiple ways, including K-12 science. In the United States, the adoption of the Next Generation Science Standards (NGSS) has provided ...

Background: Women with generalized joint hypermobility may be at increased risk of pregnancy-related pelvic girdle pain, but evidence is inconclusive. Objectives: In this prospective cohort study of 283 pregnant women in Norway, we aimed to study the association of generalized joint mobility with pelvic girdle pain, and to evaluate if pre-pregnancy body mass index was a modifier of the association. Methods: Generalized joint hypermobility was defined as a score of >= 5/9 positive tests on the Beighton score measured in early pregnancy. Primary outcome was evening pain intensity in gestation week 30, measured by a 100 mm visual analogue scale. We applied linear regression analyses to estimate age-adjusted unstandardized beta coefficients. Results: Evening pain intensity was similar among women with Beighton score >= 5/9 and women with Beighton score < 5/9 (age-adjusted mean difference 2.8 mm; 95% CI: -9.2 to 14.9 mm). Women with Beighton score >= 5/9 and pre-pregnancy body mass index >= 25 kg/m(2), reported higher evening pain than women with Beighton score < 5/9 and pre-pregnancy body mass index = 25 kg/m(2) reported higher evening pain compared to women with normal joint mobility and body mass index

Background Pelvic girdle pain is a common problem during pregnancy. For most women, the symptoms cease within the first 3–6 months of giving birth, but in some women the pain persists. In this study we investigate the sexuality and frequency of depressive symptoms in women with persistent pelvic girdle pain after childbirth and in healthy women. Methods We conducted a case–control study of women with persistent pelvic girdle pain after childbirth and a control group of healthy women. The frequency of depressive symptoms and sexuality were measured using the self-rating version of the Montgomery–Asberg Depression Rating Scale and the McCoy Female Sexuality Questionnaire. Results Forty-six women with persistent pelvic girdle pain and thirty-nine healthy women were enrolled. The frequency of depressive symptoms and the total score on female sexuality did not differ between the groups. However, pain during intercourse was more frequent (P P β= − 0,41, p Conclusion Depressive symptoms and female sexuality were similar between women with persistent pelvic girdle pain after childbirth and healthy controls. However, pain during intercourse and avoidance of sexual intercourse were more frequent among women with pelvic girdle pain.

Summary Spinal cord longitudinal axons comprise some of the longest axons in our body. However, mechanisms that drive this extra long-distance axonal growth are largely unclear. We found that ascending axons of rapidly adapting (RA) mechanoreceptors closely abut a previously undescribed population of roof plate-derived radial glial-like cells (RGLCs) in the spinal cord dorsal column, which form a network of processes enriched with growth-promoting factors. In dreher mutant mice that lack RGLCs, the lengths of ascending RA mechanoreceptor axon branches are specifically reduced, whereas their descending and collateral branches, and other dorsal column and sensory pathways, are largely unaffected. Because the number and intrinsic growth ability of RA mechanoreceptors are normal in dreher mice, our data suggest that RGLCs provide critical non-cell autonomous growth support for the ascending axons of RA mechanoreceptors. Together, our work identifies a developmental mechanism specifically required for long-range spinal cord longitudinal axons.

This review offers a state of the field examination of cookstove implementation efforts with a focus on stakeholder engagement and persistently low rates of adoption. Literature from related fields, such as sanitation and public health, indicate that perspectives in sustainable energy are narrow, and point to a new approach for sustainable energy and development engagement, one that does not solely rely on overcoming habitualized behaviors of adult women. Should stakeholder perspectives be expanded, and coupled with partnerships that include local, youth-oriented educational institutions, better uptake of efficient cooking technologies may be realized. This paper argues that youth, current and future users of cookstoves, are systematically overlooked at all points along the cookstove value chain, and that their continued exclusion from implementation efforts is to the detriment of cookstove research and practice. This paper calls for their purposeful inclusion in development efforts through collaborations with Education for Sustainable Development providers whose work is complementary to the cookstove and sustainable development communities’ aims and aspirations. This represents a new line of research in sustainable household energy, one that includes a diversity of perspectives and the inclusion of all stakeholders.

Half of all human cancers lose p53 function by missense mutations, with an unknown fraction of these containing p53 in a self-aggregated amyloid-like state. Here we show that a cell-penetrating peptide, ReACp53, designed to inhibit p53 amyloid formation, rescues p53 function in cancer cell lines and in organoids derived from high-grade serous ovarian carcinomas (HGSOC), an aggressive cancer characterized by ubiquitous p53 mutations. Rescued p53 behaves similarly to its wild-type counterpart in regulating target genes, reducing cell proliferation and increasing cell death. Intraperitoneal administration decreases tumor proliferation and shrinks xenografts invivo. Our data show the effectiveness of targeting a specific aggregation defect of p53 and its potential applicability to HGSOCs.

Improved cookstoves and solar cookers have been positioned as a socio-technical strategy for improving household health and economics, and for reducing the negative impacts on the environment associated with the combustion of residential solid fuels in low- and middle-income countries. However, adoption and sustained use of these technologies have not been realized. This critical review examines the ways in which cookstove programs use information-sharing and education-related Behavior Change Communication techniques to engage all stakeholders within a household to promote long-term cookstove usage. Findings indicate that few cookstove and solar cooker adoption studies (n = 18) report such strategies, and of those that do, rarely are men (n = 4), often the primary financial decision makers of a household, or children (n = 3), current and future users of cookstoves, purposefully engaged. Included papers report cookstove adoption metrics in a variety of ways, preventing direct comparisons between studies, by stakeholder, or behavior change strategy. Despite the lack of standardized adoption metrics, this review finds that comprehensive educational initiatives, as opposed to training or simple information provision, as part of a behavior change strategy may advance sustained use of cooking technologies. This represents an unexplored area of improved energy research.

Scientific outreach efforts traditionally involve formally trained scientists teaching the general public about the methods, significance, and excitement of science. We recently experimented with an alternative “symbiotic outreach” model that prioritizes building a reciprocal relationship between formally trained and “outsider” scientists to facilitate active two-way communication. Herein, we present the results of our outreach effort involving college students and adults with intellectual and developmental disabilities working together to make biological and multimedia art. By discussing the steps others can take to cultivate reciprocal outreach within their local communities, we hope to lower the barrier for widespread adoption of similar approaches and ultimately to decrease the gap between formally trained scientists and the general public., This Community Page article presents an alternative outreach model that prioritizes building a reciprocal relationship between scientists and outsiders, describes an example involving college students and adult artists with intellectual and developmental disabilities working together to create BioArt, and discusses how others can cultivate similar opportunities within their local communities.

The article argues that disability is a resource for revitalizing the practice of portraiture. Drawing on Tobin Siebers’s work on disability aesthetics and Rosemarie Garland-Thomson’s work on staring, the article explores how disability inflects and redefines the conventions of portraiture and how contemporary portraiture revises the photographic history of disability, a history rooted in the medicalization or enfreakment of bodily difference. After briefly surveying the history of photographic representations of variant bodies, consideration is given to the work of Diane Arbus, whose portraits continue to shape the representation of disability, and then to the work of contemporary photographer Laura Swanson, who positions her work in relation to that of Arbus. Swanson reimagines the portrait by resisting the viewer’s gaze and confronting viewers with their expectations of portraiture, their histories of looking, and their encounters with disability and difference.

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with β-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients.

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis – a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA.

In this study we present data to support the role for Cdk2ap2 in regulating self-renewal of mouse embryonic stem cells (mESCs) under permissive conditions, and cell survival during differentiation of the mESCs into terminally differentiated cell types. To understand the function of Cdk2ap2 during early development, we generated mESCs with homozygous disruption of the endogenous Cdk2ap2 locus (Cdk2ap2(tr/tr)). The Cdk2ap2(tr/tr) mESCs, when grown in a complete growth medium containing leukemia inhibitory factor (LIF), showed an early differentiation phenotype characterized by flattened colonies and a distinct intercellular boundary. We also observed downregulation of Nanog and upregulation in markers of mesoderm and endoderm differentiation, including Brachyury (T), Afp, and S100a, when compared to Wt mESCs. Cdk2ap2(tr/tr) mESCs were able to form embryoid bodies (EBs); however, those EBs were unhealthy and had an increased level of apoptosis. Furthermore, Cdk2ap2(tr/tr) mESCs were unable to form teratomas in severe combined immunodeficiency (SCID) mice. Cdk2ap2 under normal conditions has a biphasic expression, suggesting regulatory roles in early-versus-late stem cell differentiation. These data begin to add to our understanding of how Cdk2ap2 may be involved in the regulation of self-renewal of stem cells during early embryogenesis.

The potential for directed differentiation of human-induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS-derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS-derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS-derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases. Disclosure of potential conflicts of interest is found at the end of this article.

ObjectivesTo describe referred pain patterns provoked from intra-pelvic structures in women with chronic pelvic pain (CPP) persisting after childbirth with the purpose to improve diagnostics and give implications for treatment.Materials and methodsIn this descriptive and comparative study 36 parous women with CPP were recruited from a physiotherapy department waiting list and by advertisements in newspapers. A control group of 29 parous women without CPP was consecutively assessed for eligibility from a midwifery surgery. Inclusion criterion for CPP was: moderate pain in the sacral region persisting at least six months after childbirth confirmed by pelvic pain provocation tests. Exclusion criteria in groups with and without CPP were: persistent back or pelvic pain with onset prior to pregnancy, previous back surgery and positive neurological signs. Pain was provoked by palpation of 13 predetermined intra-pelvic anatomical landmarks. The referred pain distribution was expressed in pain drawings and described in pain maps and calculated referred pain areas.ResultsPain provoked by palpation of the posterior intra-pelvic landmarks was mostly referred to the sacral region and pain provoked by palpation of the ischial and pubic bones was mostly referred to the groin and pubic regions, with or without pain referred down the ipsilateral leg. The average pain distribution area provoked by palpation of all 13 anatomical landmarks was 30.3 mm² (19.2 to 53.7) in women with CPP as compared to 3.2 mm² (1.0 to 5.1) in women without CPP, p< 0.0001.ConclusionsReferred pain patterns provoked from intra-pelvic landmarks in women with CPP are consistent with sclerotomal sensory innervation. Magnification of referred pain patterns indicates allodynia and central sensitization. The results suggest that pain mapping can be used to evaluate and confirm the pain experience among women with CPP and contribute to diagnosis.

CAK1 encodes a protein kinase in Saccharomyces cerevisiae whose sole essential mitotic role is to activate the Cdc28p cyclin-dependent kinase by phosphorylation of threonine-169 in its activation loop. SMK1 encodes a sporulation-specific mitogen-activated protein (MAP) kinase homolog that is required to regulate the postmeiotic events of spore wall assembly. CAK1 was previously identified as a multicopy suppressor of a weakened smk1 mutant and shown to be required for spore wall assembly. Here we show that Smk1p, like other MAP kinases, is phosphorylated in its activation loop and that Smk1p is not activated in a cak1 missense mutant. Strains harboring a hyperactivated allele of CDC28 that is CAK1 independent and that lacks threonine-169 still require CAK1 to activate Smk1p. The data indicate that Cak1p functions upstream of Smk1p by activating a protein kinase other than Cdc28p. We also found that mutants lacking CAK1 are blocked early in meiotic development, as they show substantial delays in premeiotic DNA synthesis and defects in the expression of sporulation-specific genes, including IME1. The early meiotic role of Cak1p, like the postmeiotic role in the Smk1p pathway, is CDC28 independent. The data indicate that Cak1p activates multiple steps in meiotic development through multiple protein kinase targets.

Background: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal. Findings: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression. Conclusions: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.

In this essay, we propose that the Deleuzoguattarian rhizome offers a map and metaphor for the field of disability studies, especially as it develops outside the boundaries of a defined program or curriculum. As an example of rhizomatic growth, we discuss a series of events in the Philadelphia area in fall 2012 that focused on disability studies and disability arts and culture, including an art exhibition entitled What Can A Body Do? and a scholarly residency sponsored by the Greater Philadelphia Women’s Studies Consortium. We suggest that the art gallery offers a generative space for the growth of disability studies, disability aesthetics, and new models of access, and we emphasize the importance of cross-institutional collaboration in the development of disability studies not only as a field but as a field of energy.Keywords: rhizome, Deleuze and Guattari, art gallery, contemporary art, curator, access, audio description, multisensory, collaboration, consortium, disability aesthetics, What Can A Body Do?&nbsp

Pregnancy-related low back and pelvic pain is a worldwide problem. A large proportion of women still experience disabling daily back pain 2 years after childbirth, resulting in major changes in activities and general well-being. In spite of this, the source of pain and effective treatment are uncertain.To evaluate the short-term effects on function of a single corticosteroid injection treatment to the ischiadic spine in women with persistent pregnancy-related pelvic pain (PPPP).Thirty-six women were allocated to injection treatment with slow-release triamcinolone and lidocain or saline and lidocain, given once at the sacrospinous ligament insertion on the ischiadic spine bilaterally with follow-up at 4 weeks. Outcome measures were Disability Rating Index (DRI), self-rated functional health (SF-36), gait speed and endurance (6MWT), and strength and endurance of trunk muscles (isometric trunk extensor and flexor tests).Women in the triamcinolone group showed significantly improved DRI (p = 0.046), 6MWT (p = 0.016), and isometric trunk extensor tests (p = 0.004), as compared with the saline group. Close co-variation was shown between improved function and reduced pain intensity.Improved function was achieved among women with PPPP after a single injection treatment with slow-release corticosteroid. The effect was positively correlated to the reduced pain intensity.

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.

Pluripotent stem cells hold significant promise in regenerative medicine due to their unlimited capacity for self-renewal and potential to differentiate into any cell type of the body. In this study, we demonstrate that proper mitochondrial function is essential for proliferation of undifferentiated ESCs. Attenuating mitochondrial function under self-renewing conditions makes these cells more glycolytic-dependent, and it is associated with an increase in the mRNA reserves of Nanog, Oct4, and Sox2. In contrast, attenuating mitochondrial function during the first 7 days of differentiation results in normal repression of Oct4, Nanog, and Sox2. However, differentiation potential is compromised as revealed by abnormal transcription of multiple Hox genes. Furthermore, under differentiating conditions in which mitochondrial function is attenuated, tumorigenic cells continue to persist. Our results, therefore establish the importance of normal mitochondrial function in ESC proliferation, regulating differentiation, and preventing the emergence of tumorigenic cells during the process of differentiation.

The germ cell lineage and germ line inheritance have fascinated ­biologists for centuries. Clinically, the function of germ cells in sexually reproducing organisms is to ensure reproductive fitness and to guard against extinction. However, the consequence of abnormal germ cell development can lead to devastating outcomes, including germ cell tumors, spontaneous recurrent miscarriage, fetal demise, infant morbidity and mortality, and birth defects. To overcome abnormal germ cell development, it has been proposed that germ cells could be generated from pluripotent stem cells in vitro. The technology for creating germ cells in this manner remains theoretical. However, recent advances suggest that the field is progressing toward this goal. In particular, the identification, isolation, and characterization of the initial step in human germ cell development has recently been reported by multiple groups. Furthermore, the birth of live, healthy, fertile young has now been achieved following fertilization of murine in vitro–derived germ cells. This remarkable achievement in the mouse has stemmed from years of studies aimed at understanding the first step in murine germ cell development, the formation of primordial germ cells (PGCs). This chapter describes the events in human PGC development in vivo and how this information should instruct PGC differentiation from human pluripotent stem cells in vitro.

Pluripotent stem cells (PSCs) hold significant promise in regenerative medicine due to their unlimited capacity for self-renewal and potential to differentiate into every cell type in the body. One major barrier to the use of PSCs is their potential risk for tumor initiation following differentiation and transplantation in vivo. In the current study we sought to evaluate the role of the tumor suppressor Pten in murine PSC neoplastic progression. Using eight functional assays that have previously been used to indicate PSC adaptation or transformation, Pten null embryonic stem cells (ESCs) failed to rate as significant in five of them. Instead, our data demonstrate that the loss of Pten causes the emergence of a small number of aggressive, teratoma-initiating embryonic carcinoma cells (ECCs) during differentiation in vitro, while the remaining 90-95% of differentiated cells are non-tumorigenic. Furthermore, our data show that the mechanism by which Pten null ECCs emerge in vitro and cause tumors in vivo is through increased survival and self-renewal, due to failed repression of the transcription factor Nanog.

Randomized double blind controlled clinical trial.To evaluate the pain relief effect of locally injected corticosteroid treatment in women with long-lasting sacral low back pain with onset during pregnancy.Pregnancy-related low back pain is a global problem. Almost 1 of 10 women still experienced disabling daily back pain 2 years after childbirth with high impact on the individual, family, and society. On spite of this, the sources of pain and effective treatment are uncertain.Thirty-six women were allocated to injection treatment, with slow-release triamcinolone and lidocaine or saline and lidocaine, given at the sacrospinous ligament insertion on the ischiadic spine bilaterally with 4 weeks follow-up time. Primary outcome measure was reported pain intensity on visual analogue scale and secondary outcome measures number of pain-drawing locations and pain-provoking test results. RESULTS.: The triamcinolone treatment group had significantly reduced pain intensity, number of pain locations, and pain-provoking test results between baseline and follow-up as compared with the saline treatment group. The absolute median change of visual analogue scale score in the triamcinolone treatment group was -24 mm and in the saline group +4.5 mm (P0.05). A reduced number of pain drawing locations was reported by 16 of 18 women in the triamcinolone group as compared with 10 of 18 in the saline group (P0.05). In the triamcinolone treatment group, 17 of 18 women had an improved pain provocation test result as compared with 9 of 18 in the saline treatment group (P0.01).The anatomic region around the sacrospinous ligament insertion on the ischial spine is suggested to be one source of long-lasting sacral low back pain with onset during pregnancy. The pain was relieved by slow-release corticosteroid injection treatment to the ischial spine.

The roof plate is an organizing center in the dorsal CNS that controls specification and differentiation of adjacent neurons through secretion of the BMP and WNT signaling molecules.Lmx1a, a member of the LIM-homeodomain (LIM-HD) transcription factor family, is expressed in the roof plate and its progenitors at all axial levels of the CNS and is necessary and sufficient for roof plate formation in the spinal cord. In the anterior CNS, however, a residual roof plate develops in the absence ofLmx1a. Lmx1b, another member of the LIM-HD transcription factor family which is highly related toLmx1a, is expressed in the roof plate in the anterior CNS. AlthoughLmx1b-null mice do not show a substantial deficiency in hindbrain roof plate formation,Lmx1a/Lmx1bcompound-null mutants fail to generate hindbrain roof plate. This observation indicates that both genes act in concert to direct normal hindbrain roof plate formation. Since the requirement ofLmx1bfunction for normal isthmic organizer at the mid–hindbrain boundary complicates analysis of a distinct dorsal patterning role of this gene, we also used a conditional knock-out strategy to specifically delete dorsal midlineLmx1bexpression. Phenotypic analysis of single and compound conditional mutants confirmed overlapping roles forLmx1genes in regulating hindbrain roof plate formation and growth and also revealed roles in regulating adjacent cerebellar morphogenesis. Our data provides the first evidence of overlapping function of theLmx1genes during embryonic CNS development.

Mouse embryonic stem cells (mESCs) maintain pluripotency and indefinite self-renewal through yet to be defined molecular mechanisms. Leukemia inhibitory factor has been utilized to maintain the symmetrical self-renewal and pluripotency of mESCs in culture. It has been suggested that molecules with significant cellular effects on retinoblastoma protein (pRb) or its related pathways should have functional impact on mESC proliferation and differentiation. However, the involvement of pRb in pluripotent differentiation of mESCs has not been extensively elaborated. In this paper, we present novel experimental data indicating that Cdk2ap1 (cyclin-dependent kinase 2-associating protein 1), an inhibitor of G(1)/S transition through down-regulation of CDK2 and an essential gene for early embryonic development, confers competency for mESC differentiation. Targeted disruption of Cdk2ap1 in mESCs resulted in abrogation of leukemia inhibitory factor withdrawal-induced differentiation, along with altered pRb phosphorylation. The differentiation competency of the Cdk2ap1(-/-) mESCs was restored upon the ectopic expression of Cdk2ap1 or a nonphosphorylatable pRb mutant (mouse Ser(788) --Ala), suggesting that the CDK2AP1-mediated differentiation of mESCs was elicited through the regulation of pRb. Further analysis on mESC maintenance or differentiation-related gene expression supports the phosphorylation at serine 788 in pRb plays a significant role for the CDK2AP1-mediated differentiation of mESCs. These data clearly demonstrate that CDK2AP1 is a competency factor in the proper differentiation of mESCs by modulating the phosphorylation level of pRb. This sheds light on the role of the establishment of the proper somatic cell type cell cycle regulation for mESCs to enter into the differentiation process.

The derivation of germ cells from human embryonic stem cells (hESCs) or human induced pluripotent stem (hIPS) cells represents a desirable experimental model and potential strategy for treating infertility. In the current study, we developed a triple biomarker assay for identifying and isolating human primordial germ cells (PGCs) by first evaluating human PGC formation during the first trimester in vivo. Next, we applied this technology to characterizing in vitro derived PGCs (iPGCs) from pluripotent cells. Our results show that codifferentiation of hESCs on human fetal gonadal stromal cells significantly improves the efficiency of generating iPGCs. Furthermore, the efficiency was comparable between various pluripotent cell lines regardless of origin from the inner cell mass of human blastocysts (hESCs), or reprogramming of human skin fibroblasts (hIPS). To better characterize the iPGCs, we performed Real-time polymerase chain reaction, microarray, and bisulfite sequencing. Our results show that iPGCs at day 7 of differentiation are transcriptionally distinct from the somatic cells, expressing genes associated with pluripotency and germ cell development while repressing genes associated with somatic differentiation (specifically multiple HOX genes). Using bisulfite sequencing, we show that iPGCs initiate imprint erasure from differentially methylated imprinted regions by day 7 of differentiation. However, iPGCs derived from hIPS cells do not initiate imprint erasure as efficiently. In conclusion, our results indicate that triple positive iPGCs derived from pluripotent cells differentiated on hFGS cells correspond to committed first trimester germ cells (before 9 weeks) that have initiated the process of imprint erasure. Disclosure of potential conflicts of interest is found at the end of this article.

Human germ cell tumors are often metastatic, presumably due to distal site tumor growth by cancer stem cells. To determine whether cancer stem cells can be identified in a transplantation model of testicular germ cell tumor, we transplanted murine embryonic germ cells (EGCs) into the testis of adult severe combined immunodeficient mice. Transplantation resulted in a locally invasive solid tumor, with a cellular component that generated secondary tumors upon serial transplantation. The secondary tumors were invariably metastatic, a feature not observed in the primary tumors derived from EGCs. To characterize the differences between EGCs and the tumor-derived stem cells, we performed karyotype and microarray analysis. Our results show that generation of cancer stem cells is associated with the acquisition of nonclonal genomic rearrangements not found in the originating population. Furthermore, pretreatment of EGCs with a potent inhibitor of self-renewal, retinoic acid, prevented tumor formation and the emergence of these genetically unstable cancer stem cells. Microarray analysis revealed that EGCs and first- and second-generation cancer stem cells were highly similar; however, approximately 1,000 differentially expressed transcripts could be identified corresponding to alterations in oncogenes and genes associated with motility and development. Combined, the data suggest that the activation of oncogenic pathways in a cellular background of genetic instability, coupled with an inherent ability to self-renew, is involved in the acquisition of metastatic behavior in the cancer stem cell population of tumors derived from pluripotent cells.

Aim: The aim of this research was to study the effect of seroma on women's perception of daily functional and emotional status after surgical treatment for breast cancer. Furthermore, the experiences of the actual drainage procedure of seroma was studied. Method: The study had a prospective and comparative design. A study specific questionnaire was developed and used. Eighty-two women operated with modified radical mastectomy completed the forms. Forty-one women with seroma were compared with the 41 without seroma. The items focused on women's perceptions of daily-life situations, postoperative pain, problems with the surgical scar, preoperative information, general health, levels of anxiety, depression, psychosocial support and contact with the registered nurse in the hospital. Results: Overall the perceived emotional and functional status, pain and general health did not differ between the two groups. Women with seroma contacted the registered nurse in the hospital more frequently after hospital discharge. Most women with seroma had no or little pain and anxiety during the aspiration and found the procedure fully acceptable. Practical information concerning self-care and the aspiration procedure was considered insufficient. The return visit to the nurse for wound observation was important and provided psychosocial support. Conclusion: Seroma and its drainage is well accepted. Patients should be better informed about the possibility of a seroma and its treatment. Staff continuity and particularly the presence of a special trained nurse for wound control and for psychosocial support are much appreciated.

BACKGROUND: General joint hypermobility is estimated to affect about 10% of the population and is a prerequisite of heritable connective tissue disorders where fragile connective tissue is a prominent feature. Pregnancy induced back pain is common whereas about 10% of women still have disabling pain several years after childbirth. The pathogenesis of the pain condition is uncertain, although several risk factors are suggested including general joint hypermobility. In the present study, the possible association of peripheral joint mobility in early pregnancy on the incidence of back pain with onset during pregnancy and persisting after childbirth was explored. METHODS: A cohort of 200 pregnant women recruited from antenatal health care clinics was assessed by questionnaire and clinical examination, including measurement of passive abduction of the left fourth finger, throughout pregnancy and at 13 weeks postpartum. Comparisons were made between women with and without back pain. Statistical tests used were χ2-test, t-test, Spearman correlation and multiple logistic regression. RESULTS: In the cohort, the mean passive abduction angle of the left fourth finger increased from 40.1° in early pregnancy to 41.8° at the postpartum appointment. At the postpartum appointment, women in the back pain group had a significantly larger mean passive abduction angle of the left fourth finger of 4.4°, twice as many previous pregnancies and deliveries, and more than twice as frequent back pain in previous pregnancy, as compared with women with no persistent back pain. A similar pattern was displayed in late pregnancy. In a multiple regression analysis, the passive abduction angle of the left fourth finger in early pregnancy and the number of previous pregnancies were positively, significantly and independently associated to the incidence of back pain in late pregnancy and postpartum. CONCLUSIONS: Finger joint laxity as a reflection of constitutional weakness of connective tissue and number of previous pregnancies were associated with the development of back pain induced in pregnancy and persisting after childbirth. These factors may provide a foundation for development of targeted prevention strategies, but this have to be confirmed in future research including measurement of general joint laxity.

Saccharomyces cerevisiae mutants that fail to complete meiotic recombination are blocked by the RAD17/RAD24/MEC1 checkpoint signaling pathway in pachytene when early sporulation genes are expressed. Middle genes are not activated in checkpoint-arrested cells because the Ndt80 transcription factor is inhibited. We find that the pachytene checkpoint requires Sum1, a transcriptional repressor that recognizes a subset of Ndt80-binding sites. Mutants lacking Sum1 or Rad17 partially bypass the block to the nuclear divisions but do not form spores, while mutants lacking both Sum1 and Rad17 completely bypass the block and form morphologically normal spores. The level of Sum1 protein decreases as middle genes are expressed, and this decrease is blocked in checkpoint-arrested cells. These data suggest that Sum1 levels are regulated by the checkpoint and that progression of the meiotic divisions and spore differentiation can be differentially controlled by competition of the Sum1 repressor and Ndt80 activator for occupancy at key middle promoters.

Two organic, spin-on BARCs are in the small scale manufacturing phase -- with the goal being a 193-nm product optimized for commercialization. Chemistries of the BARCs are shown in this paper and performance of the two products relative to industry accepted needs is discussed. The thermoset BARCs, EXP98090B and EXP99001D, are prepared from hydroxy-functional, dye-attached acrylic polymers by adding an aminoplast and sulfonic acid catalyst. With select 193-nm resists, the BARCs give resolution of L/S pairs down to 0.12 micrometer. Plasma etch rates of both BARCs are comparable to those of 193-nm photoresists. Other BARC performance parameters that are discussed for the two products include: film and optical properties, conformality, simulated reflectance curves, spin-bowl compatibility, metals content, and defects.

Etv2, a master regulator of endothelial cell fate, can induce fast skeletal muscle cells to transdifferentiate into endothelial cells in the zebrafish embryo., Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly., Author Summary The endothelial cell is a specialized cell type that lines blood vessels. These cells are involved in normal cardiovascular function and become damaged in cardiovascular disease states such as atherosclerosis and stroke. We have discovered that developing muscle cells in the zebrafish embryo can be converted into endothelial cells by the expression of a transcription factor called Etv2. Etv2 normally functions during embryonic development to specify blood and blood vessels. When expressed in muscle cells, Etv2 induces the expression of genes that are normally expressed in endothelial cells; it also represses muscle gene expression. On expressing Etv2, muscle cells change shape and go on to form lumenized blood vessels that connect to the existing circulatory system and support blood flow. The Wnt and VEGF signaling pathways are required for this fate transformation. Our results suggest that muscle cells may be a viable source for the de novo generation of endothelial cells for use in transplantation therapies and they highlight signalling pathways that might be manipulated to improve the efficiency of this process in mammalian cells.

The cell intrinsic programming that regulates mammalian primordial germ cell (PGC) development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs) from mouse embryonic stem cells (ESCs). Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright) cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright) iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright) iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

Generating induced pluripotent stem cells (iPSCs) requires massive epigenome reorganization. It is unclear whether reprogramming of female human cells reactivates the inactive X chromosome (Xi), like in mouse. Here we establish that human (h)iPSCs derived from several female fibroblasts under standard culture conditions carry an Xi. Despite the lack of reactivation, the Xi undergoes defined chromatin changes, and expansion of hiPSCs can lead to partial loss of XIST RNA. These results indicate that hiPSCs are epigenetically dynamic and do not display a pristine state of X-inactivation with two active X’s as found in some female human embryonic stem cells. Furthermore, while fibroblasts are mosaic for the Xi, hiPSCs are clonal for the Xi. This non-random pattern of X chromosome inactivation in female hiPSCs, which is maintained upon differentiation, has critical implications for clinical applications and disease modeling, and could be exploited for a unique form of gene therapy for X-linked diseases.

SummaryTransgenic zebrafish embryos expressing tissue-specific green fluorescent protein (GFP) can provide an unlimited supply of primary embryonic cells. Agents that promote the differentiation of these cells may be beneficial for therapeutics. We report a high-throughput approach for screening small molecules that regulate cell differentiation using lineage-specific GFP transgenic zebrafish embryonic cells. After validating several known regulators of the differentiation of endothelial and other cell types, we performed a screen for proangiogenic molecules using undifferentiated primary cells from flk1-GFP transgenic zebrafish embryos. Cells were grown in 384-well plates with 12,128 individual small molecules, and GFP expression was analyzed by means of an automated imaging system, which allowed us to screen thousands of compounds weekly. As a result, 23 molecules were confirmed to enhance angiogenesis, and 11 of them were validated to promote the proliferation of mammalian human umbilical vascular endothelial cells and induce Flk1+ cells from murine embryonic stem cells. We demonstrated the general applicability of this strategy by analyzing additional cell lineages using zebrafish expressing GFP in pancreatic, cardiac, and dopaminergic cells.