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Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2 . We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2 . In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

Biological systems achieve precise control over ambient fluids through the self-organization of active protein structures including flagella, cilia, and cytoskeletal networks. In active structures individual proteins consume chemical energy to generate force and motion at molecular length scales. Self-organization of protein components enables the control and modulation of fluid flow fields on micron scales. The physical principles underlying the organization and control of active-matter driven fluid flows are poorly understood. Here, we apply an optically-controlled active-matter system composed of microtubule filaments and light-switchable kinesin motor proteins to analyze the emergence of persistent flow fields in a model active matter system. Using light, we form contractile microtubule networks of varying shape. We analyze the fluid flow fields generated by a wide range of microtubule network geometries and explain the resulting flow fields within a unified theoretical framework. We specifically demonstrate that the geometry of microtubule flux at the boundary of contracting microtubule networks predicts the steady-state fluid flow fields across polygonal network geometries through finite-element simulations. Our work provides a foundation for programming microscopic fluid-flows with controllable active matter and could enable the engineering of versatile and dynamic microfluidic devices.

Introduction: Migraine is a polygenic multifactorial disorder with a neuronal initiation of a cascade of neurochemical processes leading to incapacitating headaches. Headaches are generally unilateral, throbbing, 4–72 h in duration, and associated with nausea, vomiting, photophobia, and sonophobia. Chronic migraine (CM) is the presence of a headache at least 15 days per month for ≥3 months and has a high global impact on health and economy, and therapeutic guidelines are lacking. Methods: Using the Grading of Recommendations, Assessment, Development, and Evaluations system, we conducted a search in MEDLINE and Cochrane to investigate the current evidence and generate recommendations of clinical practice on the identification of risk factors and treatment of CM in adults. Results: We recommend avoiding overmedication of non-steroidal anti-inflammatory drugs (NSAIDs); ergotamine; caffeine; opioids; barbiturates; and initiating individualized prophylactic treatment with topiramate eptinezumab, galcanezumab, erenumab, fremanezumab, or botulinum toxin. We highlight the necessity of managing comorbidities initially. In the acute management, we recommend NSAIDs, triptans, lasmiditan, and gepants alone or with metoclopramide if nausea or vomiting. Non-pharmacological measures include neurostimulation. Conclusions: We have identified the risk factors and treatments available for the management of CM based on a grading system, which facilitates selection for individualized management.

Understanding how to promote organ and appendage regeneration is a key goal of regenerative medicine. The frog, Xenopus, can achieve both scar-free healing and tissue regeneration during its larval stages, although it predominantly loses these abilities during metamorphosis and adulthood. This transient regenerative capacity, alongside their close evolutionary relationship with humans, makes Xenopus an attractive model to uncover the mechanisms underlying functional regeneration. Here, we present an overview of Xenopus as a key model organism for regeneration research and highlight how studies of Xenopus have led to new insights into the mechanisms governing regeneration.

Summary While it is appreciated that reactive oxygen species (ROS) can act as second messengers in both homeostastic and stress response signaling pathways, potential roles for ROS during early vertebrate development have remained largely unexplored. Here, we show that fertilization in Xenopus embryos triggers a rapid increase in ROS levels, which oscillate with each cell division. Furthermore, we show that the fertilization-induced Ca2+ wave is necessary and sufficient to induce ROS production in activated or fertilized eggs. Using chemical inhibitors, we identified mitochondria as the major source of fertilization-induced ROS production. Inhibition of mitochondrial ROS production in early embryos results in cell-cycle arrest, in part, via ROS-dependent regulation of Cdc25C activity. This study reveals a role for oscillating ROS levels in early cell cycle regulation in Xenopus embryos., Graphical Abstract, Highlights • ROS, including hydrogen peroxide, are produced after fertilization in Xenopus • Ca2+ signaling after fertilization induces ROS production in mitochondria • Mitochondria are the major source of oscillating ROS levels • ROS regulate Cdc25C activity and the early cell cycle, Han et al. show that the fertilization-triggered calcium wave induces reactive oxygen species production from mitochondria, which oscillate with each cell division in Xenopus embryos. Moreover, they demonstrate that inhibition of mitochondrial ROS production disrupts cell cycle progression.

Thyroid dyshormonogenesis is a leading cause of congenital hypothyroidism, a highly prevalent but treatable condition. Thyroid hormone synthesis is dependent on the formation of reactive oxygen species (ROS). In humans, the primary sources for ROS production during thyroid hormone synthesis are the NADPH oxidase, DUOX1 and DUOX2. Indeed mutations inDUOX1andDUOX2have been linked with congenital hypothyroidism. Unlike humans, zebrafish has a single orthologue forDUOX1andDUOX2. In this study, we investigated the phenotypes associated with two nonsense mutant alleles of the singleduoxgene in zebrafish,sa9892andsa13017. Both alleles gave rise to readily observable phenotypes reminiscent of congenital hypothyroidism, from the larval stages through to adulthood. By using various methods to examine the external and internal phenotypes, we discovered a strong correlation between TH synthesis andduoxfunction, beginning from the early larval stage, when T4levels are already noticeably absent in the mutants. Loss of T4production resulted in growth retardation, pigmentation defects, ragged fins, thyroid hyperplasia / external goiter, and infertility. Remarkably all of these defects associated with chronic congenital hypothyroidism could be rescued with T4treatment, even when initiated when the fish had already reached adulthood. Our work suggests that these zebrafishduoxmutants may provide a powerful model to understand the aetiology of untreated and treated congenital hypothyroidism even in advance stages of development.

Fertilisation triggers embryonic development culminating with the activation of a number of highly co-ordinated and evolutionarily conserved signalling pathways, which induce and pattern the mesoderm of the developing embryo. Previous studies in invertebrates have shown that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), can act as a signalling molecule for axis specification during early development. Using a HyPer transgenicXenopus laevisline that expresses a H2O2-sensitive fluorescent protein sensor maternally, we recently found that fertilisation triggers a rapid increase in ROS production. Here we show that this increase in ROS levels is sustained throughout early embryogenesis, lasting until the tailbud stages. In addition we show that lowering ROS levels from the blastula stage through the gastrula stages via antioxidant treatments disrupts mesoderm formation. Furthermore, we show that attenuating ROS levels during the blastula / gastrula stages affects some, but not all, growth factor signalling pathways involved in mesoderm induction and patterning, including the PI3K/Akt, TGF-β/Nodal, and Wnt/β-catenin signalling pathways. These data suggest that sustained elevated ROS levels during the blastula and gastrula stages are essential for early vertebrate embryonic development, at least partly, through their roles in promoting growth factor signalling.

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices. Copyright: © 2016 Beal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

O tromboembolismo pulmonar (TEP) é uma importante causa de morbimortalidade nos pacientes submetidos a procedimentos neurocirúrgicos. O objetivo deste estudo foi apresentar um caso de trombólise intra-arterial pulmonar em pós-operatório recente de neurocirurgia. Paciente do sexo masculino, sendo submetido a clipagem de aneurisma de artéria comunicante anterior, apresentou como complicação no sétimo dia de pós-operatório, tromboembolismo pulmonar maciço, apresentando instabilidade hemodinâmica, sendo optado pela trombólise intra-arterial pulmonar com alteplase. Apresentou evolução satisfatória, sem complicações hemorrágicas, recebendo alta hospitalar. O tromboembolismo pulmonar é uma condição com alta morbi-mortalidade no pós-operatório de neurocirurgia, devendo ser a trombólise ser uma alternativa terapêutica nos casos refratários ao tratamento clínico.Pulmonary thromboembolism is a major cause of morbidity and mortality of patients undergoing neurosurgical procedures. The purpose of this study was to present a case of intra-arterial pulmonary thrombolysis in recent neurosurgery postoperative period. Male patient, undergoing neurosurgery, presented as a complication on the seventh day of postoperative massive pulmonary embolism with hemodynamic instability and intraarterial pulmonary thrombolysis with alteplase was indicated. Evolution was satisfactory without bleeding complications and patient was discharged. Pulmonary thromboembolism is a high morbidity and mortality condition at neurosurgical postoperative period and thrombolysis should be an alternative therapy in cases refractory to clinical treatment.

JUSTIFICATIVA E OBJETIVOS: O Transfusion-Related Acute Lung Injury (TRALI), é definido como um edema pulmonar não cardiogênico, relacionado à transfusão de sangue ou derivados, evoluindo com necessidade de ventilação mecânica na grande maioria dos casos. O objetivo deste estudo foi apresentar um caso de TRALI em pós-operatório imediato de neurocirurgia. RELATO DO CASO: Paciente do sexo masculino, 69 anos, sem comprometimento pulmonar prévio, foi submetido à ressecção cirúrgica de glioblastoma multiforme, apresentando complicações intra-operatórias (broncoespasmo e diminuição da saturação de oxigênio), após ter recebido plasma fresco congelado, sendo diagnosticado TRALI. O paciente foi mantido sedado, sob ventilação mecânica e monitorização hemodinâmica invasiva, com melhora progressiva do quadro, recebendo alta da unidade de terapia intensiva (UTI) no 8º dia de pós-operatório. CONCLUSÕES: O TRALI deve ser investigado nos pacientes que recebem hemoderivados e apresentam alterações pulmonares.BACKGROUND AND OBJECTIVES: The Transfusion-Related Acute Lung Injury (TRALI), is defined as noncardiogenic pulmonary edema temporally related to transfusion therapy, evolving with ventilation necessity mechanics in the great majority of the cases. This objective of this study was to present case of TRALI in the immediate postoperative of neurosurgery. CASE REPORT: We describe the case of a patient who presented broncoespasm and decreased oxygen saturation after to have received fresh-frozen plasma in the neurosurgery, who presented TRALI. The patient was submitted a invasive hemodynamic monitoring, sedation and supplemental oxygen with mechanical ventilation, with gradual improvement, leaving the intensive care unit in the eight day of postoperative. CONCLUSIONS: The TRALI must be investigated in the patients who receive transfusion therapy and present lung injury.

Embryonic development requires exquisite regulation of several essential processes, such as patterning of tissues and organs, cell fate decisions, and morphogenesis. Intriguingly, these diverse processes are controlled by only a handful of signalling pathways, and mis-regulation in one or more of these pathways may result in a variety of congenital defects and diseases. Consequently, investigating how these signalling pathways are regulated at the molecular level is essential to understanding the mechanisms underlying vertebrate embryogenesis, as well as developing treatments for human diseases. Here, we designed and performed a large-scale gain-of-function screen in Xenopus embryos aimed at identifying new regulators of MAPK/Erk, PI3K/Akt, BMP, and TGF-β/Nodal signalling pathways. Our gain-of-function screen is based on the identification of gene products that alter the phosphorylation state of key signalling molecules, which report the activation state of the pathways. In total, we have identified 20 new molecules that regulate the activity of one or more signalling pathways during early Xenopus development. This is the first time that such a functional screen has been performed, and the findings pave the way toward a more comprehensive understanding of the molecular mechanisms regulating the activity of important signalling pathways under normal and pathological conditions. © 2013 Zhang et al.

There remains a critical need for new therapeutics that promote wound healing in patients suffering from chronic skin wounds. This is, in part, due to a shortage of simple, physiologically and clinically relevant test systems for investigating candidate agents. The skin of amphibians possesses a remarkable regenerative capacity, which remains insufficiently explored for clinical purposes. Combining comparative biology with a translational medicine approach, we report the development and application of a simple ex vivo frog (Xenopus tropicalis) skin organ culture system that permits exploration of the effects of amphibian skin-derived agents on re-epithelialisation in both frog and human skin. Using this amphibian model, we identify thyrotropin-releasing hormone (TRH) as a novel stimulant of epidermal regeneration. Moving to a complementary human ex vivo wounded skin assay, we demonstrate that the effects of TRH are conserved across the amphibian-mammalian divide: TRH stimulates wound closure and formation of neo-epidermis in organ-cultured human skin, accompanied by increased keratinocyte proliferation and wound healing-associated differentiation (cytokeratin 6 expression). Thus, TRH represents a novel, clinically relevant neuroendocrine wound repair promoter that deserves further exploration. These complementary frog and human skin ex vivo assays encourage a comparative biology approach in future wound healing research so as to facilitate the rapid identification and preclinical testing of novel, evolutionarily conserved, and clinically relevant wound healing promoters.

Wound healing is essential for survival. We took advantage of the Xenopus embryo, which exhibits remarkable capacities to repair wounds quickly and efficiently, to investigate the mechanisms responsible for wound healing. Previous work has shown that injury triggers a rapid calcium response, followed by the activation of Ras homolog (Rho) family guanosine triphosphatases (GTPases), which regulate the formation and contraction of an F-actin purse string around the wound margin. How these processes are coordinated following wounding remained unclear. Here we show that inositol-trisphosphate 3-kinase B (Itpkb) via its enzymatic product inositol 1,3,4,5-tetrakisphosphate (InsP 4 ) plays an essential role during wound healing by modulating the activity of Rho family GTPases and F-actin ring assembly. Furthermore, we show that Itpkb and InsP 4 modulate the speed of the calcium wave, which propagates from the site of injury into neighboring uninjured cells. Strikingly, both overexpression of itpkb and exogenous application of InsP 4 accelerate the speed of wound closure, a finding that has potential implications in our quest to find treatments that improve wound healing in patients with acute or chronic wounds.

To estimate the effects on homicide rates of the gang truce that was brokered in El Salvador in 2012.Mathematical models based on municipal-level census, crime and gang-intelligence data were used to estimate the effect of the truce on homicide rates. One model estimated the overall effect after accounting for the linear trend and seasonality in the homicide rate. In a moderated-effect model, we investigated the relationship between the truce effect and the numbers of MS13 (Mara Salvatrucha 13) and Eighteenth-Street gang members imprisoned per 100 000 population. We then ran each of these two models with additional control variables. We compared values before the truce - 1 January 2010 to 29 February 2012 - with those after the truce - 1 March 2012 to 31 December 2013.The overall-effect models with and without additional control variables indicated a homicide rate after the truce that was significantly lower than the value before the truce, giving rate ratios of 0.55 (95% confidence interval, CI: 0.49-0.63) and 0.61 (95% CI: 0.54-0.69), respectively. For any given municipality, the effectiveness of the truce appeared to increase as the number of MS13 gang members imprisoned per 100 000 population increased. We did not observe the same significant relationship for imprisoned Eighteenth-Street gang members.In the 22 months following the establishment of a national gang truce, the homicide rate was about 40% lower than in the preceding 26 months. The truce's impact appeared particularly strong in municipalities with relatively high numbers of imprisoned MS13 gang members per 100 000 population.Estimer les effets sur le taux d'homicide de la trêve des gangs qui a été signée au Salvador en 2012.Des modèles mathématiques établis d'après les recensements municipaux et les renseignements sur les gangs et la criminalité ont été utilisés pour estimer l'effet de la trêve sur le taux d'homicide. Un modèle a permis d'en estimer l'effet global après prise en compte de la tendance linéaire et de la saisonnalité du taux d'homicide. Un modèle à effets modérés nous a permis d'étudier la relation entre l'effet de la trêve et le nombre de membres emprisonnés des gangs MS13 (Mara Salvatrucha 13) et Barrio 18 pour 100 000 habitants. Nous avons ensuite soumis chacun de ces deux modèles à des variables de contrôle supplémentaire. Nous avons comparé les valeurs d'avant la trêve – du 1er janvier 2010 au 29 février 2012 – à celles d'après la trêve – du 1er mars 2012 au 31 décembre 2013.Les modèles d'estimation de l'effet global, avec et sans variables de contrôle supplémentaire, ont indiqué un taux d'homicide après la trêve nettement inférieur aux valeurs d'avant la trêve, avec des ratios de taux respectifs de 0,55 (intervalle de confiance de 95%, IC: 0,49-0,63) et 0,61 (IC 95%: 0,54-0,69). Quelle que soit la municipalité, l'efficacité de la trêve grandissait au fur et à mesure que le nombre de membres emprisonnés du gang MS13 pour 100 000 habitants augmentait. Nous n'avons pas observé de rapport aussi significatif pour les membres emprisonnés du gang Barrio 18.Pendant les 22 mois qui ont suivi l'établissement d'une trêve nationale des gangs, le taux d'homicide était environ 40% plus faible que lors des 26 mois précédents. L'impact de la trêve était particulièrement marqué dans les municipalités où le nombre de membres emprisonnés du gang MS13 pour 100 000 habitants était relativement élevé.Estimar el impacto en las tasas de homicidio de la tregua entre bandas negociada en El Salvador en 2012.Se utilizaron modelos matemáticos basados en un censo municipal, datos sobre crímenes e inteligencia de bandas para estimar el impacto de la tregua en las tasas de homicidio. Un modelo calculó el impacto general tras tener en cuenta la tendencia lineal y la estacionalidad de las tasas de homicidio. En un modelo de efecto moderado, se investigó la relación entre el efecto de la tregua y el número de miembros de las bandas MS13 (Mara Salvatrucha 13) y Calle 18 en prisión por cada 100 000 habitantes. Posteriormente, se implementaron ambos modelos con variables de control adicionales. Se compararon los valores antes de la tregua (del 1 de enero de 2010 al 29 de febrero de 2012) y los de después de la tregua (del 1 de marzo de 2012 al 31 de diciembre de 2013).Los modelos de efecto general con y sin variables de control adicionales indicaron que la tasa de homicidio posterior a la tregua fue mucho menor que el valor antes de la misma, lo que ofrece coeficientes de la tasa del 0,55% (intervalo de confianza, IC, del 95%: 0,49-0,63) y del 0,61 (IC del 95%: 0,54-0,69), respectivamente. En todos los municipios, la eficacia de la tregua parecía aumentar a medida que aumentaba el número de miembros de la MS13 encarcelados por cada 100 000 habitantes. No se observó la misma relación significativa para los miembros de la Calle 18 encarcelados.Durante los 22 meses posteriores al establecimiento de una tregua nacional entre bandas, la tasa de homicidio fue un 40% inferior que en los 26 meses anteriores. El impacto de la tregua fue especialmente significativo en los municipios con un número relativamente alto de miembros de la MS13 encarcelados por cada 100 000 habitantes.تقدير التأثيرات على معدل جرائم القتل نتيجة الهدنة بين العصابات التي تمت إقامتها في السلفادور عام 2012.تم استخدام نماذج رياضية تستند إلى البيانات المتعلقة بتعداد السكان على مستوى البلديات والجرائم والمعلومات الاستخبارية عن العصابات لتقدير تأثير الهدنة على معدلات جرائم القتل. قام نموذج واحد بتقدير التأثير الكلي بعد تفسير الاتجاه الخطي والتغيّرات الموسمية في معدل جرائم القتل. وفي أحد النماذج ذات التأثير المتوسط، قمنا بالتحقيق في العلاقة بين تأثير الهدنة وأعداد المسجونين من أفراد عصابة "إم إس 13" (مارتا ساڤاتروتشا 13) وعصابة "الشارع الثماني عشر" لكل 100,000 من السكان. ثم قمنا بعرض كلا هذين النموذجين مع المتغيرات الضابطة الإضافية. وقمنا بمقارنة القيم الصادرة قبل إقامة الهدنة – بدءًا من يوم 1 يناير/كانون الثاني 2010 حتى يوم 29 فبراير/شباط 2012 – مع تلك القيم الصادرة بعد إقامة الهدنة – بدءًا من يوم 1 مارس/آذار 2012 حتى يوم 31 ديسمبر/كانون الأول 2013.إن النماذج ذات التأثير الكلي المزودة بالمتغيرات الضابطة الإضافية – أو غير المزودة بها – أشارت إلى أن معدل جرائم القتل التي حدثت بعد إقامة الهدنة كان أقل بكثير من قيمة معدل الجرائم قبل إقامة الهدنة، مما يدل على نسب معدلات تبلغ 0.55 (بنسبة أرجحية مقدارها ‏95%: 0.49–0.63) و0.61 (بنسبة أرجحية مقدارها 95‏%: 0.54–0.69)، على التوالي. وبالنسبة لأي من البلديات، فقد بدا أن تأثير الهدنة يزداد حيث ارتفع عدد المسجونين من عصابة "إم إس 13" لكل 100,000 من السكان. بينما لم نلحظ العلاقة ذات الدلالة الإحصائية نفسها للأفراد المسجونين من عصابة "الشارع الثماني عشر".كان معدل جرائم القتل أقل بحوالي ‏40% في الـ 22 شهرًا التالية لإقامة هدنة بين العصابات على المستوى الوطني، مما كان عليه في الـ 26 شهرًا السابقة. وبدا أن تأثير الهدنة قويًا بشكل خاص في البلديات التي تضم أعدادًا كبيرة نسبيًا من الأفراد المسجونين من عصابة "إم إس 13" لكل 100,000 من السكان.旨在估计 2012 年在萨尔瓦多促成的帮派停战协议对凶杀率的影响。.我们使用依据市级普查、犯罪和帮派情报数据制定的数学模型估计停战协议对凶杀率的影响。 在考虑凶杀率的线性趋势和季节性之后，我们使用一种模型估计了总体影响。 在调整后的影响模型中，我们研究了停战协议的成效与每 100 000 人中入狱的救世鳟鱼帮 (MS13) 和十八街黑帮成员人数之间的关系。 然后，我们采用其他控制变量分别运行了这两个模型。 我们比较了停战协议之前的数值——2010 年 1 月 1 日至 2012 年 2 月 29 日——与停战协议之后的数值——2012 年 3 月 1 日至 2013 年 12 月 31 日。.采用和不采用其他控制变量的总体效应模型表明停战协议之后的凶杀率显著低于停战协议之前，比率分别达到 0.55（95％ 置信区间，CI: 0.49–0.63） 和 0.61 (95% CI: 0.54–0.69)。 对于任何指定的直辖市来说，由于每 100 000 人中入狱的 MS13 帮成员人数的增加，停战协议的成效也随之呈现出提高。 我们没有观察到入狱的十八街黑帮成员呈现出同等显著程度的关系。.在全国帮派停战协议签订之后的 22 个月内，凶杀率相较于之前的 26 个月降低了 40% 左右。 在每 100 000 人中入狱的 MS13 帮成员人数相对较高的直辖市中，停战协议的成效尤为显著。.Дать оценку влияния перемирия между бандами, заключенного в Сальвадоре в 2012 году, на показатели числа убийств.Оценка влияния перемирия на показатели числа убийств была дана с помощью математических моделей на основе переписных данных муниципального уровня, данных о преступности и разведывательных данных о бандах. С помощью одной модели общее влияние рассчитывалось после учета линейного тренда и сезонных колебаний показателей числа убийств. В модели модифицированного влияния были изучены связи между влиянием перемирия и количеством членов банды MS13 (Мара Сальватруча 13 (Mara Salvatrucha 13)) и банды 18-й улицы (Eighteenth-Street), заключенных в тюрьму, из расчета на 100 000 жителей. Затем обе эти модели были реализованы с дополнительными контрольными переменными. Значения, относящиеся к периоду до перемирия (от 1 января 2010 года до 29 февраля 2012 года), были сравнены со значениями, относящимися к периоду после перемирия (от 1 марта 2012 года до 31 декабря 2013 года).Реализация моделей общего влияния с дополнительными контрольными переменными и без них показала, что уровни числа убийств после перемирия были значительно меньше значений до него. В результате были получены отношения рисков, равные 0,55 (95%-й доверительный интервал, ДИ: 0,49–0,63) и 0,61 (95%-й ДИ: 0,54–0,69) соответственно. Для каждого муниципального образования было выявлено, что эффективность перемирия напрямую зависела от того, сколько членов банды MS13, заключенных под стражу, приходилось на 100 000 жителей. Такая же заметная связь с количеством заключенных под стражу членов банды 18-й улицы не наблюдалась.Через 22 месяца после заключения национального перемирия между бандами показатели убийств снизились приблизительно на 40% по сравнению с предыдущими 26 месяцами. Влияние перемирия оказалось особенно сильным в муниципальных образованиях с относительно большим количеством заключенных в тюрьму членов банды MS13 (из расчета на 100 000 жителей).

Here we present a protocol for generating transgenic embryos in Xenopus laevis and Xenopus tropicalis. The method includes three steps: (1) The preparation of high-speed egg extracts, which facilitates the replacement of protamines in sperm nuclei with nucleosomes and decondenses the chromatin of sperm nuclei; (2) The isolation of sperm nuclei; and (3) The mixing of sperm nuclei, restriction enzyme, and high-speed extract in vitro, following by nuclear transplantation into unfertilized eggs to generate the transgenic embryos. This procedure generates non-mosaic transgenic embryos at high frequency and efficiency.

Fibroblast growth factor (FGF) signalling has been implicated during several phases of early embryogenesis, including the patterning of the embryonic axes, the induction and/or maintenance of several cell lineages and the coordination of morphogenetic movements. Here, we summarise our current understanding of the regulation and roles of FGF signalling during early vertebrate development.

Frog Genome The African clawed frog Xenopus tropicalis is the first amphibian to have its genome sequenced. Hellsten et al. (p. 633 , see the cover) present an analysis of a draft assembly of the genome. The genome of the frog, which is an important model system for developmental biology, encodes over 20,000 protein-coding genes, of which more than 1700 genes have identified human disease associations. Detailed comparison of the content of protein-coding genes with other tetrapods—human and chicken—reveals extensive shared synteny, occasionally spanning entire chromosomes.

Fibroblast growth factor (FGF) signalling plays a major role during early vertebrate development. It is involved in the specification of the mesoderm, control of morphogenetic movements, patterning of the anterior-posterior axis, and neural induction. In mammals, 22 FGF ligands have been identified, which can be grouped into seven subfamilies according to their sequence homology and function. We have cloned 17 fgf genes from Xenopus tropicalis and have analysed their temporal expression by RT-PCR and spatial expression by whole mount in situ hybridisation at key developmental stages. It reveals the diverse expression pattern of fgf genes during early embryonic development. Furthermore, our analysis shows the transient nature of expression of several fgfs in a number of embryonic tissues. This study constitutes the most comprehensive description of the temporal and spatial expression pattern of fgf ligands and receptors during vertebrate development to date. Developmental Dynamics 238:1467-1479, 2009. (c) 2009 Wiley-Liss, Inc.

The mechanisms that establish behavioral, cognitive, and neuroanatomical asymmetries are poorly understood. In this study, we analyze the events that regulate development of asymmetric nuclei in the dorsal forebrain. The unilateral parapineal organ has a bilateral origin, and some parapineal precursors migrate across the midline to form this left-sided nucleus. The parapineal subsequently innervates the left habenula, which derives from ventral epithalamic cells adjacent to the parapineal precursors. Ablation of cells in the left ventral epithalamus can reverse laterality in wild-type embryos and impose the direction of CNS asymmetry in embryos in which laterality is usually randomized. Unilateral modulation of Nodal activity by Lefty1 can also impose the direction of CNS laterality in embryos with bilateral expression of Nodal pathway genes. From these data, we propose that laterality is determined by a competitive interaction between the left and right epithalamus and that Nodal signaling biases the outcome of this competition.

The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus . This technique relies on the generation of transgenic lines that carry “activator” or “effector” constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter. Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype. Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus .

Signal transduction through the FGF receptor is essential for the specification of the vertebrate body plan. Blocking the FGF pathway in early Xenopus embryos inhibits mesoderm induction and results in truncation of the anterior–posterior axis. The Drosophilagene sprouty encodes an antagonist of FGF signaling, which is transcriptionally induced by the pathway, but whose molecular functions are poorly characterized. We have cloned Xenopus sprouty2 and show that it is expressed in a similar pattern to known FGFs and is dependent on the FGF/Ras/MAPK pathway for its expression. Overexpression of Xsprouty2 in both embryos and explant assays results in the inhibition of the cell movements of convergent extension. Although blocking FGF/Ras/MAPK signaling leads to an inhibition of mesodermal gene expression, these markers are unaffected by Xsprouty2, indicating that mesoderm induction and patterning occurs normally in these embryos. Finally, using Xenopus oocytes we show that Xsprouty2 is an intracellular antagonist of FGF-dependent calcium signaling. These results provide evidence for at least two distinct FGF-dependent signal transduction pathways: a Sprouty-insensitive Ras/MAPK pathway required for the transcription of most mesodermal genes, and a Sprouty-sensitive pathway required for coordination of cellular morphogenesis.

Summary: Morpholino (MO) based inhibition of translational initiation represents an attractive methodology to eliminate gene function during Xenopus development (Heasman et al., 2000). However, the degree to which a given target protein can be eliminated and the longevity of this effect during embryogenesis has not been documented. To examine the efficacy of MOs, we have used transgenic Xenopus lines that harbour known numbers of integrations of a GFP reporter under the control of the ubiquitous and highly expressed CMV promoter (Fig. 1a). In addition we have investigated the longevity of the inhibitory effect by using transgenic lines expressing GFP specifically in the lens of tadpoles. These transgenic lines represent the ideal control for the technique as the promoters are highly expressed and GFP can be easily detected by fluorescence and immunoblotting. Moreover, as GFP has no function in development, the levels of inhibition can be tested in an otherwise normal individual. Here we report that MOs are able to efficiently and specifically inhibit the translation of GFP in transgenic lines from Xenopus laevis and Xenopus tropicalis and the inhibitory effect is long-lived, lasting into the tadpole stages. genesis 30:110–113, 2001. © 2001 Wiley-Liss, Inc.

The centromere region of Saccharomyces cerevisiae chromosome III has been replaced by various DNA fragments from the centromere regions of yeast chromosomes III and XI. A 289-base pair centromere (CEN3) sequence can stabilize yeast chromosome III through mitosis and meiosis. The orientation of the centromeric fragments within chromosome III has no effect on the normal mitotic or meiotic behavior of the chromosome. The structural integrity of the centromere region in these genomic substitution strains was examined by mapping nucleolytic cleavage sites within the chromatin DNA. A nuclease-protected centromere core of 220-250 base pairs was evident in all of the genomic substitution strains. The position of the protected region is determined strictly by the centromere DNA sequence. These results indicate that the functional centromere core is contained within 220-250 base pairs of the chromatin DNA that is structurally distinct from the flanking nucleosomal chromatin.

The recently described Spred protein family has been implicated in the modulation of receptor tyrosine kinase signalling. We report the crystal structure of the Enabled/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain from Xenopus tropicalis Spred1, solved to 1.15 A resolution. This structure confirms that the Spred EVH1 adopts the pleckstrin-homology fold, with a similar secondary structure to Enabled. A translation of one of the peptide-binding groove β-strands narrows this groove, whilst one end of the groove shows structural flexibility. We propose that Spred1 will bind peptides that are less proline-rich than other EVH1 domains, with conformational changes indicating an induced fit.

Background Trigeminal nerves consist of ophthalmic, maxillary, and mandibular branches that project to distinct regions of the facial epidermis. In Xenopus embryos, the mandibular branch of the trigeminal nerve extends toward and innervates the cement gland in the anterior facial epithelium. The cement gland has previously been proposed to provide a short-range chemoattractive signal to promote target innervation by mandibular trigeminal axons. Brain derived neurotrophic factor, BDNF is known to stimulate axon outgrowth and branching. The goal of this study is to determine whether BDNF functions as the proposed target recognition signal in the Xenopus cement gland. Results We found that the cement gland is enriched in BDNF mRNA transcripts compared to the other neurotrophins NT3 and NT4 during mandibular trigeminal nerve innervation. BDNF knockdown in Xenopus embryos or specifically in cement glands resulted in the failure of mandibular trigeminal axons to arborise or grow into the cement gland. BDNF expressed ectodermal grafts, when positioned in place of the cement gland, promoted local trigeminal axon arborisation in vivo. Conclusion BDNF is necessary locally to promote end stage target innervation of trigeminal axons in vivo, suggesting that BDNF functions as a short-range signal that stimulates mandibular trigeminal axon arborisation and growth into the cement gland.