Mutations in GDF5 Reveal a Key Residue Mediating BMP Inhibition by NOGGIN

Signaling output of bone morphogenetic proteins (BMPs) is determined by two sets of opposing interactions, one with heterotetrameric complexes of cell surface receptors, the other with secreted antagonists that act as ligand traps. We identified two mutations (N445K,T) in patients with multiple synostosis syndrome (SYM1) in the BMP–related ligand GDF5. Functional studies of both mutants in chicken micromass culture demonstrated a gain of function caused by a resistance to the BMP–inhibitor NOGGIN and an altered signaling effect. Residue N445, situated within overlapping receptor and antagonist interfaces, is highly conserved among the BMP family with the exception of BMP9 and BMP10, in which it is substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to NOGGIN and show a high chondrogenic activity. Ectopic expression of BMP9 or the GDF5 mutants resulted in massive induction of cartilage in an in vivo chick model presumably by bypassing the feedback inhibition imposed by endogenous NOGGIN. Swapping residues at the mutation site alone was not sufficient to render Bmp9 NOG-sensitive; however, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. In conclusion, we show a new mechanism for abnormal joint development that interferes with a naturally occurring regulatory mechanism of BMP signaling

Innovations, challenges, and minimal information for standardization of humanized mice.

Mice xenotransplanted with human cells and/or expressing human gene products (also known as "humanized mice") recapitulate the human evolutionary specialization and diversity of genotypic and phenotypic traits. These models can provide a relevant in vivo context for understanding of human-specific physiology and pathologies. Humanized mice have advanced toward mainstream preclinical models and are now at the forefront of biomedical research. Here, we considered innovations and challenges regarding the reconstitution of human immunity and human tissues, modeling of human infections and cancer, and the use of humanized mice for testing drugs or regenerative therapy products. As the number of publications exploring different facets of humanized mouse models has steadily increased in past years, it is becoming evident that standardized reporting is needed in the field. Therefore, an international community-driven resource called "Minimal Information for Standardization of Humanized Mice" (MISHUM) has been created for the purpose of enhancing rigor and reproducibility of studies in the field. Within MISHUM, we propose comprehensive guidelines for reporting critical information generated using humanized mice

The mortality after release from incarceration consortium (MARIC): Protocol for a multi-national, individual participant data meta-analysis

Introduction More than 30 million adults are released from incarceration globally each year. Many experience complex physical and mental health problems, and are at markedly increased risk of preventable mortality. Despite this, evidence regarding the global epidemiology of mortality following release from incarceration is insufficient to inform the development of targeted, evidence-based responses. Many previous studies have suffered from inadequate power and poor precision, and even large studies have limited capacity to disaggregate data by specific causes of death, sub-populations or time since release to answer questions of clinical and public health relevance. Objectives To comprehensively document the incidence, timing, causes and risk factors for mortality in adults released from prison. Methods We created the Mortality After Release from Incarceration Consortium (MARIC), a multi-disciplinary collaboration representing 29 cohorts of adults who have experienced incarceration from 11 countries. Findings across cohorts will be analysed using a two-step, individual participant data meta-analysis methodology. Results The combined sample includes 1,337,993 individuals (89% male), with 75,795 deaths recorded over 9,191,393 person-years of follow-up. Conclusions The consortium represents an important advancement in the field, bringing international attention to this problem. It will provide internationally relevant evidence to guide policymakers and clinicians in reducing preventable deaths in this marginalized population

The force of the sign. Terrorism as a symbolic phenomenon

Seit der radikalen Änderung der weltpolitischen Lage durch das Selbstmordattentat islamistischer Terroristen auf die Supermacht U.S.A. am 11.9.01 versucht die Weltöffentlichkeit, dem Phänomen des Terrorismus durch die verschiedensten Erklärungsstrategien Herr zu werden. Man führt den Kampf der Kulturen oder Religionen an, verweist auf die Ausbeutung der Peripherie des kapitalistischen Weltsystems durch den Hegemon U.S.A. oder pathologisiert bzw. dämonisiert die Taten der Terroristen. Uns scheinen diese Erklärungsstrategien den Machtkonstellationen der postmodernen Gesellschaften nicht gerecht zu werden, da sie ein zentrales soziales Phänomen der vergangenen Jahrzehnte nicht beachten: das Unbehagen an der pluralistischen Gesellschaft und der konsensuell-repräsentativen Demokratie. Um dieses Unbehagen angemessen beschreiben zu können, wollen wir uns ihm aus einer zeichentheoretischen Perspektive nähern, die soziale Formationen nicht durch (kooperative) Arbeit determiniert sieht, sondern durch die Instanz des Codes. Für Jean Baudrillard ist die Wahl des Ziels der beiden Türme bedeutend, die sich wechselseitig reflektieren und das System nach allen Seiten hin abschließen. Mit ihnen wurde das neuralgische Zentrum des Systems getroffen, das sich auf einem binären Code gründet. Die Twintowers bedeuteten nicht nur das Ende jedweder originalen Referenz, sondern auch den Abschluss des Bezeichneten durch die Wiederholung des Zeichens. Der Code führt eine symbolische Verteilung der gesellschaftlichen Körper durch und zielt auf eine möglichst genaue Übereinstimmung der Gemeinschaft mit sich selbst gemäß eines arithmetischen und geometrischen Kalküls. Die integrative Kraft dieses auf dem Identitätsprinzip basierenden Systems scheint immer häufiger nicht mehr in der Lage zu sein, das negative Potential antagonistischer Strategien binden zu können, die in Form von zivilem Ungehorsam, Politik von Minoritäten oder terroristischen Akten die symbolische Ordnung der westlichen Gesellschaften unterhöhlen. In diesem Tagungsband diskutieren wir das Ausmaß der Krise der konsensuellen Demokratie und ihrer Institutionen. Die Aufsätze behandeln folgende Themen: • Die Krise der pluralistischen Gesellschaft: Wie verständlich ist das kulturelle, politische und religiöse Unbehagen am westlichen Wertekanon? • Die Funktion der Medien in der konsensuellen Demokratie; die gesellschaftliche Wirkung der medialen Reproduktion terroristischer Akte; das Bild des Terrorismus in der Populärkultur • Ästhetischer Terror: Die Vernichtung von Sinn und Bedeutung in der zeitgenössischen Kunst und Kunsttheorie • Die politische Logik des Terrorismus (Ziele, Bedingungen u. Legitimität des Terrorismus in Abgrenzung zu alternativen politischen Ausdrucksformen wie z.B. dem zivilen Ungehorsam; die Bedeutung des terroristischen Opfertodes) • Geschichte des Terrorismus (speziell der Terrorismus der RAF) • Terror als irrationale Gewalt (der ontologische Status des Terrorismus)Since the radical changes in international politics caused by the suicide attacks on the USA by Islamic terrorists on the 11th of September in 2001, the world public tries to grasp the phenomenon of „Terrorism“ through different explanatory strategies. These include referring to the clash of cultures and of religions, to the exploitation of the periphery by the capitalist system incarnated by the USA, or pathologizing as well as demonizing the deeds of the terrorists. All these explanatory attempts do not seem to do justice to the actual constellations of power in postmodern societies, because they do not take a widespread social phenomenon of the past decades in consideration: the discomfort with the pluralistic society and with the concurrent-representative democracy. In order to describe this discomfort adequately we would like to approach the phenomenon from a semiotic perspective which does not consider social formations as determined by cooperative work but rather by the authority of the code. For Jean Baudrillard the target was meaningful since the two towers not only reflect each other but also completely close the system. With them the neuralgic center which is based upon the binary code was severely hit. The twin towers not only meant the end of every original reference but also the completion of the signified by the reduplication of the sign. The code conducts a symbolic distribution of the social bodies and aims at an as accurate as possible identification of the community with itself corresponding to an arithmetic and geometric calculus. The system which is based upon the principle of identity does not seem to have the ability to integrate antagonist energies properly. So these antagonist powers undermine the symbolic order of western societies in terms of civil disobedience, politics of minorities or terroristic acts. In this conference transcript we discuss the degree of crisis of consensual democracy and ist institutions. The essays cover the following topics: • The crisis of the pluralistic society: How comprehensible and justified is the cultural, political, and religious discomfort with western values? • The function of mass media in democratic systems: What is the impact of the reproduction of terrorist acts on society? How does popular culture deal with terrorism? • Aesthetic terror: the destruction of sense and meaning in contemporary arts and art theory • The political logic of terrorism (aims, legitimacy of terrorism as opposed to other forms of opposition like civil disobedience; the meaning of the terrorist self-sacrifice death) • History of terrorism (e.g. The German terrorism of the RAF) • Terrorism as an irrational power (ontological status of terrorism

Mammalian MCM Loading in Late-G1 Coincides with Rb Hyperphosphorylation and the Transition to Post-Transcriptional Control of Progression into S-Phase

BACKGROUND: Control of the onset of DNA synthesis in mammalian cells requires the coordinated assembly and activation of the pre-Replication Complex. In order to understand the regulatory events controlling preRC dynamics, we have investigated how the timing of preRC assembly relates temporally to other biochemical events governing progress into S-phase. METHODOLOGY/PRINCIPAL FINDING: In murine and Chinese hamster (CHO) cells released from quiescence, the loading of the replicative MCM helicase onto chromatin occurs in the final 3-4 hrs of G(1). Cdc45 and PCNA, both of which are required for G(1)-S transit, bind to chromatin at the G(1)-S transition or even earlier in G(1), when MCMs load. An RNA polymerase II inhibitor (DRB) was added to synchronized murine keratinocytes to show that they are no longer dependent on new mRNA synthesis 3-4 hrs prior to S-phase entry, which is also true for CHO and human cells. Further, CHO cells can progress into S-phase on time, and complete S-phase, under conditions where new mRNA synthesis is significantly compromised, and such mRNA suppression causes no adverse effects on preRC dynamics prior to, or during, S-phase progression. Even more intriguing, hyperphosphorylation of Rb coincides with the start of MCM loading and, paradoxically, with the time in late-G(1) when de novo mRNA synthesis is no longer rate limiting for progression into S-phase. CONCLUSIONS/SIGNIFICANCE: MCM, Cdc45, and PCNA loading, and the subsequent transit through G(1)-S, do not depend on concurrent new mRNA synthesis. These results indicate that mammalian cells pass through a distinct transition in late-G(1) at which time Rb becomes hyperphosphorylated and MCM loading commences, but that after this transition the control of MCM, Cdc45, and PCNA loading and the onset of DNA replication are regulated at the post-transcriptional level

The reference site collaborative network of the european innovation partnership on active and healthy ageing

Seventy four Reference Sites of the European Innovation Partnership on Active and Healthy Ageing (EIP on AHA) have been recognised by the European Commission in 2016 for their commitment to excellence in investing and scaling up innovative solutions for active and healthy ageing. The Reference Site Collaborative Network (RSCN) brings together the EIP on AHA Reference Sites awarded by the European Commission, and Candidate Reference Sites into a single forum. The overarching goals are to promote cooperation, share and transfer good practice and solutions in the development and scaling up of health and care strategies, policies and service delivery models, while at the same time supporting the action groups in their work. The RSCN aspires to be recognized by the EU Commission as the principal forum and authority representing all EIP on AHA Reference Sites. The RSCN will contribute to achieve the goals of the EIP on AHA by improving health and care outcomes for citizens across Europe, and the development of sustainable economic growth and the creation of jobs

Molecular biological investigations to the pathogenesis of the skeletal malformations SYNS1 and BDA2 in the BMP-signalling pathway

Der BMP-Signalweg nimmt eine essentielle Rolle im Bereich der Musterbildung, der Organogenese, des Wachstums und der Homöostase des Skeletts ein. Dabei ist ein fein abgestimmtes Zusammenspiel von Expressionsort, -zeit und -menge sowie die Proteininteraktion der einzelnen Komponenten im BMP-Signalweg entscheidend, da bereits geringe Abweichungen während des komplexen Bildungsprozesses zu Fehlbildungen führen. Ziel dieser Arbeit war die Aufklärung der Pathogenese zweier Handfehlbildungen, BDA2 und SYNS1, die nach genetischen Analysen mit Mutationen betreffend der sezernierten Signalmoleküle BMP2 bzw. GDF5 assoziiert sind. Beide Proteine sind Mitglieder der TGFβ- Superfamilie und zeigen ein z.T. überlappendes Expressionsprofil während der embryonalen Extremitätenentwicklung in der Gelenksregion und im Perichondrium. Die dort verlaufenden Vorgänge sind besonders kritisch für die Etablierung eines korrekt ausgebildeten Gelenks und der Determinierung der Phalangen. Für die GDF5 Mutationen N445T und N445K, die mit SYNS1 assoziiert und in der überlappenden Proteininteraktionsoberfläche mit Nog und BMPR1B lokalisiert sind, konnte in vitro eine Insensitivität gegenüber dem BMP-Antagonisten Nog nachgewiesen werden. Dies resultiert in vitro als auch in vivo in einer verstärkten chondrogenen Aktivität, die zusätzlich die endogene negative Feedback-Regulation durch Nog umgeht. Die Position N445 ist unter den BMPs hochkonserviert, jedoch tragen BMP9 und BMP10 natürlicherweise dort ein Lysin und sind wie die GDF5 Mutanten Nog-insensitiv. Während ein einfacher Aminosäureaustausch von Lysin mit Arginin keine Nog-Sensitivität in Bmp9 induziert, konnte durch gezielte Mutagenese an zwei weiteren potentiellen Interaktionsstellen eine nahezu vollständige Nog-Sensitivität vermittelt werden. Zusätzlich zeigten zu GDF5 N445T analoge Mutationen in BMP2/4/7 keine Veränderung der Nog-Sensitivität. Daher kann dem Asparagin eine sehr wichtige Funktion bei der Interaktion mit Nog zugesprochen werden, jedoch ist diese im Gegensatz zu GDF5 bei weiteren Vertretern der BMPs weniger essentiell. Diese Erkenntnisse können, im Hinblick auf klinische Anwendungen im Bereich der regenerativen Medizin, beim Design von BMPs der 2. Generation (Superagonists) genutzt werden, um eine effektivere Therapie mit geringeren Proteinkonzentrationen zu ermöglichen. Für die Mikroduplikation 110 kb downstream von BMP2 konnte mit Hilfe eines transgenen Mausmodels eine extremitätenspezifische Reportergenaktivität ermittelt werden. Das resultierende Expressionspattern von LacZ überlappt mit endogener Bmp2 Expression, was auf die Anwesenheit eines Extremitäten-spezifischen Bmp2-Enhancer in der duplizierten Region hindeutet. Darüber hinaus konnte die Position der cis regulatorischen Sequenz auf ca. 1 kb eingegrenzt werden. Diese Ergebnisse haben einen zusätzlichen Pathogenesemechanismus der BDA2 aufgedeckt, der daraufhindeutet, dass durch die Duplikation eines Enhancer die Expression von BMP2 in der Extremität verändert und das Signalisierungsgleichgewicht zwischen BMPR1A und BMPR1B verschoben wird. Beide funktionellen Untersuchungen zur Genotyp-Phänotyp Korrelation untermauern die hochsensible Regulation des BMP-Signalwegs und bieten grundlegende Einblicke in die aufeinander abgestimmten Proteininteraktionen, die sich überschneidende Signalkaskaden während Entwicklungsprozesse ermöglichen.BMP-signalling plays throughout the whole embryonic development and in the adult organism an essential role in pattern formation, organogenesis, growth and homeostasis of the skeleton. Especially during the limb development the fine-tuning of a dose dependent as well as spatially and temporally controlled expression pattern in combination with defined protein interactions between the members of the BMP-signalling pathway is crucial. Only small changes in these complex processes can result in skeletal abnormalities. This PhD thesis is devoted to clarify the pathogenesis of two skeletal malformations, BDA2 and SYNS1, which are associated with mutations concerning the secreted signalling molecules BMP2 and GDF5, respectively. Both proteins are members of the TGFβ- superfamily and show during limb development an overlapping expression pattern in the joint region as well as in the perichondrium. Patterning and differentiation processes at these locations are critical for establishing correct developed joints and phalanges. The GDF5 mutations N445T and N445K, which are associated with SYNS1, are situated in the overlapping receptor (BMPR1B) and antagonist (NOG) interface. Functional in vitro analysis of the GDF5 variants revealed a Nog-insensitivity and a change of signalling mode. Both effects result in vivo and in vitro in an enhanced protein activity supported by circumvented negative feedback loop of Nog. Interestingly, the residue N445 is highly conserved among the BMP-family except of BMP9 and BMP10 in which it is naturally substituted with lysine. Like the mutant GDF5, both BMPs are insensitive to Nog and show a high chondrogenic activity. While a simple exchange of arginine with lysine doesn’t induce Nog-sensitivity, successive introduction of two additional substitutions imparted high to total sensitivity on customized variants of Bmp9. Furthermore, homologous mutations to GDF5 N445T in BMP2/4/7 didn’t lead to Nog-insensitivity in these BMPs. Taken together, these results suggest a critical role of the asparagine in the BMP-NOG interaction face, which is less essential in BMPs than in GDF5. Out of this, these findings are useful for the design of second generation BMPs (superagonists), which can improve the effectivity of BMPs in low dosages for clinical applications in regenerative medicine. The microduplication 110 kb downstream of BMP2, which is associated with BDA2, contains evolutionary highly conserved sequences. By using a transgenic mouse model it was shown that this duplicated sequence is able to drive limb-specific reporter gene expression. The almost complete overlap of LacZ with endogenous Bmp2 expression indicates that a limb specific BMP2 enhancer is located within the duplicated sequence. Furthermore, the location of the cis-regulatory sequence was narrowed down to a ~1 kb fragment. These results reveal an additional pathogenesis mechanism of BDA2 which suggest an altered BMP2 expression through duplication of the enhancer resulting in a misbalanced BMPR1A and BMPR1B signaling in the developing limb. Both functional studies in this work concerning genotype-phenotype correlation emphasize the sensible regulation of the BMP pathway in the limb. Furthermore, they provide fundamental insights into the fine tuned protein interactions which make overlapping signalling cascades possible

Host-Pathogen Interactions of Mycoplasma mycoides in Caprine and Bovine Precision-Cut Lung Slices (PCLS) Models.

Respiratory infections caused by mycoplasma species in ruminants lead to considerable economic losses. Two important ruminant pathogens are Mycoplasma mycoides subsp. Mycoides (Mmm), the aetiological agent of contagious bovine pleuropneumonia and Mycoplasma mycoides subsp. capri (Mmc), which causes pneumonia, mastitis, arthritis, keratitis, and septicemia in goats. We established precision cut lung slices (PCLS) infection model for Mmm and Mmc to study host-pathogen interactions. We monitored infection over time using immunohistological analysis and electron microscopy. Moreover, infection burden was monitored by plating and quantitative real-time PCR. Results were compared with lungs from experimentally infected goats and cattle. Lungs from healthy goats and cattle were also included as controls. PCLS remained viable for up to two weeks. Both subspecies adhered to ciliated cells. However, the titer of Mmm in caprine PCLS decreased over time, indicating species specificity of Mmm. Mmc showed higher tropism to sub-bronchiolar tissue in caprine PCLS, which increased in a time-dependent manner. Moreover, Mmc was abundantly observed on pulmonary endothelial cells, indicating partially, how it causes systemic disease. Tissue destruction upon prolonged infection of slices was comparable to the in vivo samples. Therefore, PCLS represents a novel ex vivo model to study host-pathogen interaction in livestock mycoplasma