Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro

Mammalian embryogenesis requires intricate interactions between embryonic and extraembryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combined mouse embryonic stem cells (ESCs) and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional scaffold to generate structures whose morphogenesis is markedly similar to that of natural embryos. By using genetically modified stem cells and specific inhibitors, we show that embryogenesis of ESC- and TSC-derived embryos—ETS-embryos—depends on cross-talk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extraembryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell types resemble those of natural embryos.We are grateful to the Wellcome Trust and ERC for supporting this work. S.E.H. and C.K. are both supported by Biotechnology and Biological Sciences Research Council doctoral training partnership studentships. B.S. is supported by the International Research Fellowship Program 2214/A from Scientific and Technological Research Council of Turkey, and M.Z.-G. by the Wellcome Trust. S.E.H. served as an intern in the Cambridge, UK, office of Science/AAAS

Вплив трьох спортивних ігор з фізичного виховання на фізичну підготовленість студентів чоловічої статі

Background and Study Aim. Few controlled studies have been conducted on the effect of sports games as a physical education (PE) course on the health-related fitness of university students. The aim of the study was to determine whether three sports in a PE course will help improve the health-related fitness of male university students. Material and Methods. Students from two universities participated in the study, with one university acting as a control group. There were two PE courses which students registered for: a football and volleyball (FVG, n=169) course and a badminton (BG, n=97) course. The students received basic training drills and competed with one another. The duration of the activities was 50 minutes sessions for eight weeks. The following tests were taken before and after eight weeks: Cooper test, sit-and-reach test, 60-s curl test, standing long jump test, and body mass index. Paired t-tests were used to compare the baseline and post data of each group. The Welch t-test, ANCOVA, and analysis of gains scores were used to compare each of the PE groups to the control group. ANCOVA was used to account for baseline differences, while analysis of gains scores was used whenever ANCOVA could not be used. The Vargha-Delaney Effect Size (VD) and the Common Language Effect Size (CLES) were used to determine the effect sizes for the Welch t-tests and ANCOVA, respectively. Significant changes were set at p ≤ 0.05 and VD ≤ 42% or VD ≥ 58%, or if p ≤ 0.05 and CLES ≥ 58 %. Results.  Both PE groups showed significant improvements in all the measured fitness parameters except body composition. Moreover, the measured parameters of the control group reduced after eight weeks. Conclusion. The study shows evidence that PE courses can serve male universities in improving their health-related fitness. Moreover, students who do not participate in a PE course may be at risk of losing their fitness.Предпосылки и цель исследования. Было проведено мало контролируемых исследований о влиянии спортивных игр в качестве курса по физическому воспитанию (PE) на состояние здоровья студентов университетов. Цель исследования состояла в том, чтобы определить, помогут ли три вида спорта на курсе физического воспитания улучшить состояние здоровья студентов мужского пола.Материал и методы. В исследовании приняли участие студенты из двух университетов, один из которых выступал в качестве контрольной группы. Были два курса физическоого воспитания, на которые зарегистрировались студенты: курс по футболу и волейболу (FVG, n = 169) и курс по бадминтону (BG, n = 97). Студенты проходили базовые тренировки и соревновались друг с другом. Продолжительность занятий составляла 50 минут сеансов по восемь недель. Следующие тесты были взяты до и после восьми недель: тест Купера, тест гибкости, 60-секундный тест сгибания, тест прыжка в длину с места и индекс массы тела. Парные t-тесты использовались для сравнения базовых и пост-данных каждой группы. Для сравнения каждой из групп PE с контрольной группой использовались t-критерий Уэлча, ANCOVA и анализ результатов. ANCOVA использовалась для учета базовых различий, в то время как анализ показателей выигрышей использовался всякий раз, когда ANCOVA нельзя было использовать. Размер эффекта Варга-Делани (VD) и Размер эффекта общего языка (CLES) использовались для определения размеров эффекта для t-тестов Уэлча и ANCOVA, соответственно. Значительные изменения были установлены при p ≤ 0,05 и VD ≤ 42% или VD ≥ 58%, или если p ≤ 0,05 и CLES ≥ 58%.Результаты. Обе группы PE показали значительное улучшение всех измеренных параметров физической формы, кроме состава тела. Более того, измеренные параметры контрольной группы снизились через восемь недель.Выводы. Исследование показывает, что курсы PE могут помочь студентам улучшить свою физическую форму. Кроме того, студенты, которые не участвуют в курсе PE, могут подвергаться риску потерять свою физическую форму.Передумови та мета дослідження. Було проведено мало контрольованих досліджень про вплив спортивних ігор в якості курсу з фізичного виховання (PE) на стан здоров'я студентів університетів. Мета дослідження полягала в тому, щоб визначити, чи допоможуть три види спорту на курсі фізічного виховання поліпшити стан здоров'я студентів чоловічої статі.Матеріал і методи. У дослідженні взяли участь студенти з двох університетів, один з яких виступав в якості контрольної групи. Були два курси фізічного виховання, на які зареєструвалися студенти: курс по футболу і волейболу (FVG, n = 169) і курс з бадмінтону (BG, n = 97). Студенти проходили базові тренування і змагалися один з одним. Тривалість занять складала 50 хвилин сеансів по вісім тижнів. Наступні тести були взяті до і після восьми тижнів: тест Купера, тест гнучкості, 60-секундний тест згинання, тест стрибка в довжину з місця та індекс маси тіла. Парні t-тести використовувалися для порівняння базових і пост-даних кожної групи. Для порівняння кожної з груп PE з контрольною групою використовувалися t-критерій Уелча, ANCOVA і аналіз результатів. ANCOVA використовувалася для обліку базових відмінностей, в той час як аналіз показників виграшів використовувався щоразу, коли ANCOVA не можна було використовувати. Розмір ефекту Варга-Делані (VD) і розмір ефекту спільної мови (CLES) використовувалися для визначення розмірів ефекту для t-тестів Уелча і ANCOVA, відповідно. Значні зміни були встановлені при p ≤ 0,05 і VD ≤ 42% або VD ≥ 58%, або якщо p ≤ 0,05 і CLES ≥ 58%.Результати. Обидві групи PE показали значне поліпшення всіх виміряних параметрів фізичної форми, крім складу тіла. Більш того, виміряні параметри контрольної групи знизилися через вісім тижнів.Висновки. Дослідження показує, що курси PE можуть допомогти студентам покращити свою фізичну форму. Крім того, студенти, які не беруть участі в курсі PE, можуть піддаватися ризику втратити свою фізичну форму

Association between a rare SNP in the second intron of human Agouti related protein gene and increased BMI

<p>Abstract</p> <p>Background</p> <p>The agouti related protein (AGRP) is an endogenous antagonist of the melanocortin 4 receptor and is one of the most potent orexigenic factors. The aim of the present study was to assess the genetic variability of <it>AGRP </it>gene and investigate whether the previously reported SNP rs5030980 and the rs11575892, a SNP that so far has not been studied with respect to obesity is associated with increased body mass index (BMI).</p> <p>Methods</p> <p>We determined the complete sequence of the <it>AGRP </it>gene and upstream promoter region in 95 patients with severe obesity (BMI > 35 kg/m²). Three polymorphisms were identified: silent mutation c.123G>A (rs34123523) in the second exon, non-synonymous mutation c.199G>A (rs5030980) and c.131-42C>T (rs11575892) located in the second intron. We further screened rs11575892 in a selected group of 1135 and rs5030980 in group of 789 participants from the Genome Database of Latvian Population and Latvian State Research Program Database.</p> <p>Results</p> <p>The CT heterozygotes of rs11575892 had significantly higher mean BMI value (p = 0.027). After adjustment for age, gender and other significant non-genetic factors (presence of diseases), the BMI levels remained significantly higher in carriers of the rs11575892 T allele (p = 0.001). The adjusted mean BMI value of CC genotype was 27.92 ± 1.01 kg/m²(mean, SE) as compared to 30.97 ± 1.03 kg/m²for the CT genotype. No association was found between rs5030980 and BMI.</p> <p>Conclusion</p> <p>This study presents an association of rare allele of <it>AGRP </it>polymorphism in heterozygous state with increased BMI. The possible functional effects of this polymorphism are unclear but may relate to splicing defects.</p

Localized induction of gene expression in embryonic stem cell aggregates using holographic optical tweezers to create biochemical gradients

Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses

Human embryo polarization requires PLC signaling to mediate trophectoderm specification

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8–16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos

Mapping European Association of Urology Guideline Practice Across Europe: An Audit of Androgen Deprivation Therapy Use Before Prostate Cancer Surgery in 6598 Cases in 187 Hospitals Across 31 European Countries

Background: Evidence-practice gaps exist in urology. We previously surveyed European Association of Urology (EAU) guidelines for strong recommendations underpinned by high-certainty evidence that impact patient experience for which practice variations were suspected. The recommendation “Do not offer neoadjuvant androgen deprivation therapy (ADT) before surgery for patients with prostate cancer” was prioritised for further investigation. ADT before surgery is neither clinically effective nor cost effective and has serious side effects. The first step in improving implementation problems is to understand their extent. A clear picture of practice regarding ADT before surgery across Europe is not available. Objective: To assess current ADT use before prostate cancer surgery in Europe. Design, setting, and participants: This was an observational cross-sectional study. We retrospectively audited recent ADT practices in a multicentre international setting. We used nonprobability purposive sampling, aiming for breadth in terms of low- versus high-volume, academic, versus community and public versus private centres. Outcome measurements and statistical analysis: Our primary outcome was adherence to the ADT recommendation. Descriptive statistics and a multilevel model were used to investigate differences between countries across different factors (volume, centre type, and funding type). Subgroup analyses were performed for patients with low, intermediate, and high risk, and for those with locally advanced prostate cancer. We also collected reasons for nonadherence. Results and limitations: We included 6598 patients with prostate cancer from 187 hospitals in 31 countries from January 1, 2017 to May 1, 2020. Overall, nonadherence was 2%, (range 0–32%). Most of the variability was found in the high-risk subgroup, for which nonadherence was 4% (range 0–43%). Reasons for nonadherence included attempts to improve oncological outcomes or preoperative tumour parameters; attempts to control the cancer because of long waiting lists; and patient preference (changing one's mind from radiotherapy to surgery after neoadjuvant ADT had commenced or feeling that the side effects were intolerable). Although we purposively sampled for variety within countries (public/private, academic/community, high/low-volume), a selection bias toward centres with awareness of guidelines is possible, so adherence rates may be overestimated. Conclusions: EAU guidelines recommend against ADT use before prostate cancer surgery, yet some guideline-discordant ADT use remains at the cost of patient experience and an additional payer and provider burden. Strategies towards discontinuation of inappropriate preoperative ADT use should be pursued. Patient summary: Androgen deprivation therapy (ADT) is sometimes used in men with prostate cancer who will not benefit from it. ADT causes side effects such as weight gain and emotional changes and increases the risk of cardiovascular disease, diabetes, and osteoporosis. Guidelines strongly recommend that men opting for surgery should not receive ADT, but it is unclear how well the guidance is followed. We asked urologists across Europe how patients in their institutions were treated over the past few years. Most do not use ADT before surgery, but this still happens in some places. More research is needed to help doctors to stop using ADT in patients who will not benefit from it

In vitro generation of mouse polarized embryo-like structures from embryonic and trophoblast stem cells

Mammalian embryogenesis requires the coordination of embryonic and extra-embryonic tissues to enable implantation into the uterus and post-implantation development to establish the body plan. Mouse embryonic stem cells (ESCs) are a useful tool for studying pluripotent embryonic tissue in vitro. However, they cannot undertake correct embryogenesis alone. Many attempts to model the early embryo in vitro involve the aggregation of ESCs into spheroids of variable size and cell number that undertake germ-layer specification but fail to recapitulate the characteristic architecture and arrangement of tissues of the early embryo. Here, we describe a protocol to generate the first embryo-like structures by directing the assembly of mouse ESCs and extra-embryonic trophoblast stem cells (TSCs) in a 3D extracellular matrix (ECM) into structures we call ‘polarized embryo-like structures’. By establishing the medium and culture conditions needed to support the growth of both stem cell types simultaneously, embryonic architecture is generated within 4 d of co-culture. This protocol can be performed by those proficient in standard ESC culture techniques and can be used in developmental studies to investigate the interactions between embryonic and extra-embryonic tissues during mammalian development.We are grateful to the Wellcome Trust for the Senior Research fellowship (grant no. 098287/Z/12/Z) and for a European Research Council grant (code: 669198) awarded to M.Z.-G. to fund this work. We are also grateful for the BBSRC DTP studentship that supports S.E.H. and to the Scientific and Technological Research Council of Turkey, which supports B.S.

Effectiveness of breeding selection for grain quality in common bean.

he aims of this study were to investigate the genetic variability and the genotype × environment interaction for quality and yield traits in common bean (Phaseolus vulgaris L.), to evaluate the degree of informativeness of the evaluations of grain quality in only one environment, to estimate genetic parameters for grain quality traits, and to select lines with superior grain quality. We evaluated 81 carioca common bean lines in preliminary line trials in several environments for nutritional, technological, and commercial quality and selected the 20 superior lines, which were evaluated in validation trials in nine environments. Individual and combined ANOVAs were performed for all the traits. Correlations were estimated between Fe and Zn concentrations and yield; adaptability and phenotypic stability were analyzed; and superior genotypes were selected based on the Mulamba & Mock index. It is possible to increase the Fe, Zn, and crude protein concentrations and reduce cooking time; however, increasing crude fiber is a challenge. Preliminary evaluation of the quality traits in only one environment was effective and sufficient for selection of genotypes superior in Fe concentration, crude fiber, crude protein, and cooking time; and genetic gains can be obtained from selection for these traits. Genetic and phenotypic correlations were observed between Fe and Zn concentrations. The lines CNFC 16627, CNFC 16518, CNFC 16602, CNFC 16615, and CNFC 16520 are superior based on the selection index and are recommended for breeding for grain quality in carioca common bean