Academic literature on the topic 'MOBT relaxase'

Integrative mobilizable elements (IMEs) are widespread but very poorly studied integrated elements that can excise and hijack the transfer apparatus of co-resident conjugative elements to promote their own spreading. Sixty-four putative IMEs, harboring closely related mobilization and recombination modules, were found in 14 Streptococcus species and in Staphylococcus aureus. Fifty-three are integrated into the origin of transfer (oriT) of a host integrative conjugative element (ICE), encoding a MobT relaxase and belonging to three distant families: ICESt3, Tn916, and ICE6013. The others are integrated into an unrelated IME or in chromosomal sites. After labeling by an antibiotic resistance gene, the conjugative transfer of one of these IMEs (named IME_oriTs) and its host ICE was measured. Although the IME is integrated in an ICE, it does not transfer as a part of the host ICE (no cis-mobilization). The IME excises and transfers separately from the ICE (without impacting its transfer rate) using its own relaxase, distantly related to all known MobT relaxases, and integrates in the oriT of the ICE after transfer. Overall, IME_oriTs use MobT-encoding ICEs both as hosts and as helpers for conjugative transfer. As half of them carry lsa(C), they actively participate in the dissemination of lincosamide–streptogramin A–pleuromutilin resistance among Firmicutes.

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOBV family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOBV relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOBV histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria.

ABSTRACTThe plasmid R1162 (RSF1010) encodes a primase essential for its replication. This primase makes up the C-terminal part of MobA, a multifunctional protein with the relaxase as a separate N-terminal domain. The primase is also translated separately as the protein RepB′. Here, we map two signals for type IV secretion onto the recently solved structure of RepB′. One signal is located internally within RepB′ and consists of a long α-helix and an adjacent disordered region rich in arginines. The second signal is made up of the same α-helix and a second, arginine-rich region at the C-terminal end of the protein. Successive arginine-to-alanine substitutions revealed that either signal can be utilized by the type IV secretion complex of the plasmid R751. The internal signal also enables conjugal transfer when linked to the relaxase part of MobA. Both signals are similar to those previously identified for type IV secretion substrates in the Vir system ofAgrobacterium tumefaciens. Moreover, the C-terminal arginine-rich segment of RepB′ has been shown to be secreted by Vir. However, with R751, the signals require MobB, an R1162-encoded accessory protein active in conjugal transfer. The results of two-hybrid assays revealed that MobB interacts, via its membrane-associated domain, with the R751 plasmid coupling protein TraG. In addition, MobB interacts with a region of MobA just outside the RepB′ domain. Therefore, MobB is likely an adaptor that is essential for recognition of the primase-associated signals by the R751 secretion machinery.IMPORTANCEFor most plasmids, type IV secretion is an intrinsic part of the mechanism for conjugal transfer. Protein relaxases, bound to the 5′ end of the transferring strand, are mobilized into recipient cells by the type IV pathway. In this work, we identify and characterize two signals for secretion in the primase domain of MobA, the relaxase of the IncQ plasmid R1162 (RSF1010). We also show that the adaptor protein MobB is required for engagement of these signals with the R751 coupling protein TraG. These results clarify the location and properties of secretion signals active during the conjugal transfer of plasmid DNA.

Abstract Relaxases of the MOBH family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 domain contains two putative metal-binding motifs, an HD domain motif and an alternative 3H motif. TraI, encoded within the gonococcal genetic island of Neisseria gonorrhoeae, is the prototype of the MOBH family. SAXS experiments showed that TraI_2 and TraI_2_C form globular structures separated by an extended middle domain. The TraI_2 domain cleaves oriT-ssDNA in a site-specific Mn2+ or Co2+ dependent manner. The minimal oriT encompasses 50 nucleotides, requires an inverted repeat 3′ of the nic-site and several nucleotides around nic for efficient cleavage. Surprisingly, no stable covalent relaxase-DNA intermediate was observed. Mutagenesis of conserved tyrosines showed that cleavage was abolished in the Y212A mutant, whereas the Y212F and Y212H mutants retained residual activity. The HD and the alternative 3H motifs were essential for cleavage and the HD domain residues D162 and D267 for metal ion binding. We propose that the active site binds two metal ions, one in a high-affinity and one in a low-affinity site.

ABSTRACT Conjugative transfer from Clostridium symbiosum to enterococci of Tn1549, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (oriT) in the element. Transcription analysis of Tn1549 indicated that orf29, orf28, orfz, and orf27 were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to orf29-orf30 of Tn1549 was mobilized in Escherichia coli recA::RP4::Δnic provided that orf28 and orf29 were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in trans, but a cis-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp oriT was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp oriT fragment. The minimal oriT contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOBp superfamily. The nick site was identified within oriT by an oligonucleotide cleavage assay. Closely related oriTs linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn1549 is a member of a MOBp clade. Interestingly, the Tn1549-derived constructs were mobilized by RP4 in E. coli, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn1549 to be mobilized by a heterologous transfer system.

ABSTRACT We characterized the regions involved in replication and mobilization of the 40-kb plasmid pNZ4000, encoding exopolysaccharide (EPS) production in Lactococcus lactis NIZO B40. The plasmid contains four highly conserved replication regions with homologous rep genes (repB1, repB2,repB3, and repB4) that belong to the lactococcal theta replicon family. Subcloning of each replicon individually showed that all are functional and compatible in L. lactis. Plasmid pNZ4000 and genetically labeled derivatives could be transferred to different L. lactis strains by conjugation, and pNZ4000 was shown to be a mobilization plasmid. Two regions involved in mobilization were identified near two of the replicons; both included an oriT sequence rich in inverted repeats. Conjugative mobilization of the nonmobilizable plasmid pNZ124 was promoted by either one of these oriT sequences, demonstrating their functionality. One oriT sequence was followed by a mobA gene, coding for atrans-acting protein, which increased the frequency of conjugative transfer 100-fold. The predicted MobA protein and theoriT sequences show protein and nucleotide similarity, respectively, with the relaxase and with the inverted repeat andnic site of the oriT from the Escherichia coli plasmid R64. The presence on pNZ4000 of four functional replicons, two oriT sequences, and several insertion sequence-like elements strongly suggests that this EPS plasmid is a naturally occurring cointegrate.

Les génomes bactériens évoluent principalement grâce au transfert horizontal de gènes. La conjugaison bactérienne en est un des mécanismes majeurs. Elle est notamment médiée par les éléments intégratifs et conjugatifs (ICE). En plus de leur transfert, les ICE codent d'autres fonctions conférant à leur hôte un avantage adaptatif, comme par exemple des résistances aux antibiotiques dont la diffusion est un enjeu majeur en santé publique. Il est donc nécessaire de comprendre comment les ICE se transfèrent si l'on veut limiter leur dissémination. Le transfert d'un ICE d'une cellule donatrice vers une cellule réceptrice implique son excision du chromosome, son transfert puis son intégration dans les génomes des deux cellules partenaires. Les données de la littérature révèlent que l'initiation de ce transfert est médiée par un complexe nucléoprotéique appelé relaxosome, dont la protéine clé est la relaxase, une transestérase codée par l'élément. Le rôle de la relaxase est d'effectuer une coupure simple brin sur l'ADN de l'ICE au niveau d'un site conservé, appelé nic. Ce clivage libère une extrémité 3'OH libre, servant d'amorce pour initier la réplication en cercle roulant. Le complexe ADN-relaxase est alors dirigé vers le pore de conjugaison. Au cours de ma thèse j'ai étudié un ICE modèle, ICESt3 de Streptococcus thermophilus qui appartient à la superfamille ICESt3/Tn916/ICEBs1, très répandue chez les Firmicutes. Ces ICE possèdent une relaxase non canonique, appartenant à la famille MOBT, apparentée aux initiateurs de réplication à cercle roulant de la famille Rep_trans. L'objectif de ma thèse était d'élucider le fonctionnement de la relaxase RelSt3 afin de décrypter les mécanismes moléculaires d'initiation du transfert conjugatif médié par une relaxase MOBT. Mes recherches ont conduit à l'identification du site de liaison de RelSt3 sur l'origine de transfert (oriT) d'ICESt3. Ce site, appelé bind, a pour originalité d'être distant du site nic, ce qui n'est pas le cas des autres familles de relaxases. RelSt3 présente un domaine HTH à son extrémité N-terminal. J'ai montré que ce domaine est requis pour la fixation de RelSt3 sur le site bind, et important pour son activité catalytique. Des tests de conjugaison ont démontré que ce domaine HTH est crucial pour le transfert conjugatif d'ICESt3. Des prédictions structurales de ce domaine en complexe avec l'ADN ont conduit à l'identification de l'interface d'interaction avec le site bind, confirmée par mutagénèse dirigée. J'ai également démontré que RelSt3 présente une activité de coupure-religature et qu'elle se fixe de façon covalente sur l'extrémité 5' du brin clivé, démontrant ainsi que cette enzyme participe aux étapes initiale et terminale de la conjugaison. Dans la littérature, il a été démontré que les relaxases interagissent fréquemment avec d'autres protéines accessoires, codées par l'ICE ou la bactérie hôte pour former le relaxosome. Le deuxième objectif de ma thèse était d'identifier des partenaires de RelSt3. L'analogie avec ICEBs1 chez Bacillus subtilis a permis d'identifier deux protéines candidates OrfL et OrfM codées par ICESt3, ainsi qu'une hélicase cellulaire, probablement impliquée dans la réplication en cercle roulant, nommée PcrA. Une caractérisation de ces protéines candidates a été effectuée en utilisant des approches biochimiques et biophysiques. Le réseau d'interaction entre l'ensemble de ces protéines a été dressé en utilisant des approches in vitro, ainsi que l'approche double hybride in vivo. Ces données nous permettent d'avoir un premier aperçu des constituants du relaxasome d'ICESt3. J'ai par ailleurs montré que OrfL et OrfM stimulent l'activité catalytique de RelSt3 in vitro, et qu'elles sont toutes les deux essentielles à la conjugaison d'ICESt3.Ce travail nous apporte une meilleure compréhension des mécanismes moléculaires en jeu lors de la conjugaison d'un ICE pilotée par une relaxase de la famille MOBT
Bacterial genomes evolve mainly through horizontal gene transfer. Bacterial conjugation is one of the major mechanisms for these transfers. Conjugation is mediated by integrative and conjugative elements (ICE). In addition to their transfer function, ICEs encode other functions that may provide an adaptive advantage to their host, such as resistance to antibiotics whose dissemination is a major public health issue. It is therefore necessary to understand how ICEs are transferred in order to limit their dissemination.The transfer of an ICE from a donor cell to a recipient cell requires its excision from the chromosome, its transfer from one cell to the other and then its integration into the genomes of the two partner cells. According to the literature, the initiation of ICE transfer is mediated by a nucleoprotein complex called relaxosome, whose key protein is the relaxase, a transesterase encoded by the element. The role of the relaxase is to perform a single-stranded cleavage on the DNA of the ICE at a conserved site, called nic. This cleavage releases a free 3'OH end, used as a primer to initiate rolling circle replication. The DNA-relaxase complex is then driven to the conjugation pore.During my PhD thesis, I studied ICESt3 from Streptococcus thermophilus which belongs to the ICESt3/Tn916/ICEBs1 superfamily, widespread among Firmicutes. These ICEs encode a non-canonical relaxase belonging to the MOBT family, which is related to the rolling circle replication initiators of the Rep_trans family. The general objective of my thesis was to elucidate the function of the RelSt3 relaxase in order to decipher the molecular mechanisms of initiation of conjugative transfer mediated by a MOBT relaxase.My work led to the identification of the RelSt3 binding site on ICESt3 origin of transfer (oriT). This site, called bind, is peculiar in that it is distant from the nic site, which is not the case for other relaxase families. RelSt3 possesses an HTH domain at its N-terminus. I have shown that this domain is required for the binding of RelSt3 to its bind site, and that it is important for its catalytic activity. Conjugation assays demonstrated that this HTH domain is crucial for the conjugative transfer of ICESt3. Structural predictions of the HTH domain in complex with DNA led to the identification of the interaction interface with the bind site, confirmed by mutagenesis. I also demonstrated that RelSt3 exhibits a nicking-closing activity and that it covalently binds to the 5' end of the cleaved strand, demonstrating that this enzyme participates in both initial and final steps of conjugation.In the literature, it has been shown that relaxases interact frequently with other accessory proteins, encoded by the ICE or by the host bacteria, participating in relaxosome formation. The second objective of my thesis was to identify RelSt3 partners. Comparisons with available data on ICEBs1 from Bacillus subtilis allowed to identify two candidate proteins, OrfL and OrfM, that may belong to the relaxosome of ICESt3, as well as a cellular helicase, PcrA , probably involved in the rolling circle replication. A characterization of these proteins was performed using biochemical and biophysical approaches. The interaction network between all of these proteins was established using in vitro approaches, as well as with the in vivo two-hybrid approach. These data provide a first insight into the components of the ICESt3 relaxasome. I also showed that OrfL and OrfM stimulate the catalytic activity of RelSt3 in vitro, and that they are both essential for ICESt3 conjugation.This work lead to a better understanding of the molecular mechanisms required during the conjugation of an ICE driven by a MOBT family relaxase

MobM relaxase from the promiscuous antibiotic resistance plasmid pMV158 is a prototype of the Mob_Pre/MOBV family of relaxases, the major family of relaxases found in Staphylococcus aureus. Staphylococcal infections cause the highest number of lethal cases among antibiotic-resistant bacterial infections. Relaxases initiate the conjugative DNA transfer, a major route for the antibiotic resistance acquisition in bacteria, by nicking their substrate DNA through formation of a covalent DNA-relaxase adduct and terminate the transfer in the recipient cells by rejoining ends of the linearized plasmid. MobM forms a DNA-histidine adduct, unique to MOBV relaxases, instead of a DNA-tyrosine adduct, thus representing a distinct category of relaxases with specialization towards the transfer of short mobile genetic elements in Gram-positive pathogenic bacteria. MobM overall fold resembles the fold of other structurally characterized relaxases, although some important structural differences are present. Molecular basis for the MobM processing of plasmid origin of transfer and active site mechanism are described herein.
La relaxasa MobM del promiscuo plásmido de resistencia a antibióticos pMV158 es un prototipo de la familia Mob_Pre/MOBV de relaxasas, la mayor familia de relaxasas se encuentran en Staphylococcu aureus. Las infecciones por estafilococos causan el mayor número de casos mortales entre las infecciones bacterianas resistentes a los antibióticos. Relaxases iniciar la transferencia conjugativa de ADN, una ruta el más frecuente para la adquisición de resistencia a antibióticos por bacterias, por mellar su ADN sustrato mediante la formación de un aducto covalente de ADN-relaxasa y terminan la transferencia en las células receptoras por reincorporarse extremos del plásmido linealizado. MobM forma un aducto de ADN-histidina, único para MOBV relaxases, en lugar de un aducto de ADN-tirosina, lo que representa una categoría distinta de relaxases con especialización hacia la transferencia de elementos genéticos móviles cortos en bacterias patógenas Gram-positivas. MobM estructura general se asemeja a la de otras veces relaxases caracterizan estructuralmente, aunque algunas diferencias estructurales importantes están presentes. Base molecular para el procesamiento de origen del plásmido por MobM y mecanismo de sitio activo se describe en esta thesis.

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniera en Biotecnología Molecular.
El océano comprende el 71% de la superficie de la Tierra, participando en el control del clima, y proveyendo más del 50% del oxígeno disponible en la atmósfera. Las comunidades microbianas que habitan los ambientes marinos se caracterizan por ser determinantes en la producción primaria, además de ser diversas en sus funciones y distribución, siendo fundamentales en la mantención de los ciclos biogeoquímicos. Éstas poseen distintas estrategias para habitar estos ambientes, ya que en estos existen variaciones en los factores abióticos, tales como temperatura, disponibilidad de oxígeno o salinidad, que determinan cambios a nivel biótico. El dinamismo de los ambientes marinos favorece el intercambio de información genética mediado por la transferencia horizontal de genes (HGT). Está descrito que la conjugación es el mecanismo que posee una mayor tasa de ocurrencia en estos ambientes, por lo que estudiar los elementos genéticos móviles (EGM) conjugativos resulta necesario para comprender que genes son potencialmente transferidos. Este proceso se inicia cuando la enzima relaxasa, codificada por el EGM identifica el origen de transferencia (oriT) en el ADN del elemento a transferir, y realiza el corte con el que se inicia la movilización del elemento conjugativo a la célula receptora por medio del sistema de secreción de tipo IV (T4SS). Esta misma relaxasa es la que empalma el ADN transferido. Lo particular de esta enzima, es que es un elemento ubicuo para los elementos conjugativos, lo que la convierte junto a los genes que la codifican (genes mob) en marcadores de movilidad génica por conjugación, y por tanto en un sistema de clasificación de estos elementos. Se ha descrito que los genes transferidos en plásmidos movilizables o conjugativos son capaces de conferir capacidades adaptativas a determinados microorganismos, tales como resistencia a antibióticos o a metales pesados, mientras que a nivel comunitario su efecto aún requiere de mayor estudio. En este seminario de título se planteó la búsqueda e identificación de relaxasas y genes mob, tanto in sílico como in situ, en muestras marinas de la región de Valparaíso. In sílico, se identificaron 28 proteínas con dominios funcionales descritos para relaxasas mediante la utilización de modelos ocultos de Markov (HMM) en un metagenoma obtenido desde el proyecto “TARA Oceans”, correspondiente a una estación ubicada frente a las costas de la región central de Chile, realizando además una cuantificación de los genes mob correspondientes a las proteínas identificadas, obteniendo que más del 70% de las lecturas reclutadas respondían a sólo dos familias de relaxasas (MOBP y MOBH). Para el estudio in situ se analizaron muestras marinas colectadas de una zona intermareal en Montemar, región de Valparaíso, en los meses de enero, marzo y julio de 2018. Estas muestras se trataron utilizando distintos protocolos de extracción de ADN, diferenciando ADN total y ADN plasmidial. Se evaluó la presencia de genes codificantes para relaxasas en los distintos tratamientos de las muestras mediante DPMT (Degenerate Primer Mob Typing) donde se logró identificar la presencia de la familia MOBQu mediante esta técnica. Posteriormente se realizó una cuantificación del gen codificante para esta familia mediante q-PCR, obteniendo que la muestra extraída con un kit comercial para extracción de ADN plasmidial se encontraba enriquecida en estos genes. Con estos resultados podemos demostrar que estas estrategias permitieron la identificación de relaxasas y los genes que las codifican en sistema marino, y así inferir la presencia de elementos conjugativos en estos.
The ocean comprises 71% of the Earth's surface, participating in climate control, and providing more than 50% of the oxygen available in the atmosphere. The microbial communities that inhabit marine environments are characterized by being determinant in primary production, diverse in their functions and distribution, and fundamental in the maintenance of biogeochemical cycles. These communities have different strategies to inhabit these environments, since in these there are variations in the abiotic factors, such as temperature, availability of oxygen or salinity, which determine changes at the biotic level. The dynamism of marine environments favors the exchange of genetic information by horizontal gene transfer (HGT). It is reported that conjugation is the mechanism that has a higher rate of occurrence in these environments, so studying the conjugative mobile genetic elements (EGM) is necessary to understand which genes are potentially transferred. This process is initiated when the relaxase enzyme, encoded by the EGM, identifies the origin of transfer (oriT) in the DNA of the element to be transferred, and performs the cut with which the mobilization of the conjugative element to the recipient cell is initiated through the Type IV secretion system (T4SS). This same relaxase enzyme is the one that splices the transferred DNA. A particular issue about this enzyme is that it is a ubiquitous element for the conjugative elements, which converts it together with the genes that code it (mob genes) into markers of gene mobility by conjugation, and therefore in a classification system of these elements. It has been reported that genes transferred in mobilizable or conjugative plasmids are able to confer adaptive capacities to certain microorganisms, such as resistance to antibiotics or heavy metals, while at the community level their effect still requires further V-ix study. In this work we proposed the search and identification of relaxases and mob genes, both in silico and in situ, in marine samples from the Valparaíso region. In silico, 28 proteins with functional domains described for relaxases were identified by using hidden Markov models (HMM) in a metagenome obtained from the "TARA Oceans" project, corresponding to a station located off the coasts of the central region of Chile, also carrying out a quantification of the mob genes corresponding to the identified proteins, obtaining that more than 70% of the readings recruited responded to only two families of relaxases (MOBP and MOBH). For the in situ study, marine samples collected from an intertidal zone in Montemar, Valparaíso region, in the months of January, March and July 2018 were analyzed. These samples were subjected to different protocols for total DNA and plasmid DNA extraction, evaluating the presence of genes coding for relaxases in the different treatments of the samples by means of DPMT (Degenerate Primer Mob Typing) where it was possible to identify the presence of the MOBQu family. A quantification of the gene coding for this family was performed by q-PCR, obtaining that the sample extracted with a commercial kit for plasmid DNA extraction was enriched in these genes. With these results we can demonstrate that these strategies allowed the identification of relaxases and the genes that codify them in the marine system, and thus infer the presence of conjugative elements in them.

The place of neuromuscular blocking agents in the intensive care unit (ICU) has changed markedly over the last 20 years. Originally regarded as a mainstay of the process of ‘sedation’, they are now only used for specific indications. The principal disadvantage is probably the difficulty in neurological assessment when a muscle relaxant is used coupled with the increased risk of awareness, because inadequate sedation will be masked. Of the available agents, the intermediate acting ones are the most popular. The degree of relaxation can be readily controlled and they have few side effects. In the presence of renal and/or hepatic disease atracurium or cisatracurium are preferred. Succinylcholine is only used for securing the airway due to its very rapid onset of action. Rocuronium given in a higher dose also possesses a rapid onset in situations when succinylcholine might be contraindicated. When using a muscle relaxant, its effect should always be monitored with a simple train of four pattern of stimulation from a hand-held nerve stimulator. This will ensure that an adequate and not excessive block is secured. If a more rapid reversal is required then a dose of neostigmine with glycopyrrolate may be used. Alternatively, if rocuronium is the relaxant in use then the new agent sugammadex is effective.

Abstract The relationship of any finale to its companion movements is partly one of similarity, partly one of difference. If it is totally similar, it loses its raison deter as a separate movement; if it is totally different, it violates the broad principle of unity or (if one prefers) coherence. In a conventional three-movement or four-movement plan its relationship to the first movement is crucial for two reasons: the two movements are both fast (a factor that makes their direct comparison appropriate), and they occupy strategically equivalent (though, as we saw, very different) positions framing the rest of the work To a lesser extent, a finale has to position itself visavis the interior movements, and in particular the minuet, scherzo, or similar movement in a four-movement cycle. Its relationship to the minuet (etc.) is naturally most conspicuous when the two movements are adjacent: that is, when the minuet comes third rather than second, as it does most often from Haydn onwards until the late nineteenth century.

The ductus arteriosus and foramen ovale were described by Galen without understanding their functions. His beliefs in soul localization and spiritization within the left ventricle established religious pneumatology which became a theological need in the Middle Ages. Pulmonary transit was recognized by Servetus and Colombo after the Reformation around 1550. This prompted Harvey’s full understanding of the fetal circulation. Botallo did not describe the ductus arteriosus, but in 1564 redescribed the foramen ovale, making his way into the Nomina Anatomica by mistake. Most authors of the 19th and 20th centuries believed ductal patency to be passive, and postnatal closure to be an active process, explained by mechanical theories. After the discovery of prostaglandins by Bergstrom and Vane, Coceani proved that ductal patency is maintained by the relaxant action of prostaglandins.

This chapter looks at how Croatia's transition from communism into the European embrace has produced tremendous narrative rewriting. This in turn relaxes some of the most established historical canons of the twentieth century and creates a political environment fertile for mnemonic challenges, disruptions, and revisions. Here, Croatia's narratives about its past have become “islands of memory.” The memories of the Holocaust exist, contained within the remembrance practices of the ever-dwindling Jewish community, but isolated from the larger narrative arc of Croatia's World War II. All of this revisionism has happened not out of spite, but as a result of the EU's own practices of remembrance, especially its reductionist interpretation of the twentieth century as an era of two totalitarianisms, equal in their criminal nature.

Sighing is both performative and vital activity, and exemplifies the role of ‘primordial affectivity’ in the organism’s co-creativity with its environment. Emerging from the organism’s ‘cares’, transforming the atmosphere and the affect that initiated it, the sigh is a striking instance of distributed cognition, an action reaching through ancient respiratory processes to the most deliberate forms of self-care. Premodern psychology understood the sigh as an attempt to free the circulation of vital and animal spirits from blockage caused by the overheating of imaginative and estimative faculties when obsessed by the image of a loved object. Contemporary science similarly sees the chief physiological action of the sigh, the opening of air spaces in the lungs, as dynamically engaged with affective experience. In the domain of psychoanalysis, the sigh is a transitional phenomena; it buys time and gives us the time to open up to something new. The sigh relaxes constriction, opening the throat and enabling speech. Hence its vital importance in amorous verse. ‘Le Sigh’ proposes that sighing is the template for the concluding couplet of Shakespeare’s sonnet form. Its innovation is to give us the breathing room to bear our care-full lives.

Deep learning models are data driven. For example, the most popular convolutional neural network (CNN) model used for image classification or object detection requires large labeled databases for training to achieve competitive performances. This requirement is not difficult to be satisfied in the visible domain since there are lots of labeled video and image databases available nowadays. However, given the less popularity of infrared (IR) camera, the availability of labeled infrared videos or image databases is limited. Therefore, training deep learning models in infrared domain is still challenging. In this chapter, we applied the pix2pix generative adversarial network (Pix2Pix GAN) and cycle-consistent GAN (Cycle GAN) models to convert visible videos to infrared videos. The Pix2Pix GAN model requires visible-infrared image pairs for training while the Cycle GAN relaxes this constraint and requires only unpaired images from both domains. We applied the two models to an open-source database where visible and infrared videos provided by the signal multimedia and telecommunications laboratory at the Federal University of Rio de Janeiro. We evaluated conversion results by performance metrics including Inception Score (IS), Frechet Inception Distance (FID) and Kernel Inception Distance (KID). Our experiments suggest that cycle-consistent GAN is more effective than pix2pix GAN for generating IR images from optical images.

The kava plant, Piper methysticum Forst. f., is an attractive shrub in the pepper family, Piperaceae (figure 12.1). Known by various names in tropical Pacific, such as yagona, kava, kava kava, ’awa, seka, and sakau, it is propagated vegetatively, as are most of the traditional crops in the region. Kava has been used for many centuries to produce psychoactive preparations. Its active principles, several lipidlike substances known as kavalactones, are concentrated in the rootstock and roots. These psychoactive chemicals are ingested traditionally by Pacific islanders as cold-water infusions of chewed, ground, pounded, or otherwise macerated kava stumps and roots. Mind-altering kava preparations are, or once were, imbibed in a wide range of Pacific Ocean societies. These include peoples living in some lowland areas on the large Melanesian island of New Guinea in the western Pacific to very isolated islands such as those in Polynesian Hawai’i, 7,000 kilometers to the northeast (figure 12.2). Beyond this widespread local use in the tropical Pacific, utilization of kava in parts of Europe as a plant source for medicinal preparations has a relatively lengthy history. In Europe it has been used as a sedative, tranquilizer, muscle relaxant, relief from menopausal symptoms, and treatment for urinary tract and bladder ailments (Lebot et al. 1999). Over the past decade, there has been rapidly increasing interest in kava well beyond the areas of traditional use among Pacific Islanders (figure 12.3). This includes a huge surge in the use of kava products in Europe, North America, Australia, and elsewhere. Within the past 3 to 5 years there has been widespread recognition of its potential to emerge as a mainstream herbal product. Modern cultivation and use of kava in the Pacific has significantly expanded in some traditional use areas such as Vanuatu, Fiji, Tonga, Samoa, and Pohnpei. There are also significant signs of rejuvenated interest in kava cultivation in some traditional areas of use where it had been abandoned because of depopulation, political prohibition, or zealous missionary denunciation. Increasing use and cultivation of kava on these Pacific islands has been stimulated by local consumption rates and rising demand for commercial export.

Optical wavelength converters will provide both flexibility and efficiency to future WDM networks due to the ability to allocate wavelengths on a link-to-link basis which in turn relaxes requirements to management and wavelength precision [1]. This has lead to a considerable attention to wavelength converters. Although different converters have been proposed the most promising seems to be the interferometric wavelength converters that use semiconductor optical amplifiers (SOAs) in interferometric structures such as Michelson (MI) and Mach-Zehnder (MZI) configurations [2]. These converters can be operated at high speed [3], they have low chirp [4,5] as well as regenerative capabilities [6] and are wavelength independent [5].

Abstract This model estimates the probability of completing a product development process over time. The Design Structure Matrix (DSM) framework is used to capture the information dependencies between tasks using the concepts of Probability of Change and Impact. The model incorporates a stochastic element that represents the likelihood of changes resulting in task iterations. The model captures the dynamic behavior of a product development process formed by a combination of parallel, serial and coupled tasks. The model relaxes the assumption that coupled tasks take place in a complete parallel or serial iteration. It can be used to compare the development time of the project for different task sequences and overlapping degrees. This tool allows for identification of the leverage points in the system, providing information about the most effective way to reduce development time. This project was a joint effort with a Hewlett-Packard division, and the observations and practical application presented are based on this field experience.

Current wind farm layout optimization research focuses on advancing optimization methods. The research includes the assumption that a continuous piece of land is readily available. In reality, wind farm development projects rely on the permission of landowners for success. When a viable wind farm site location is identified, local residents are approached for permission to build turbines on their land, typically in exchange for monetary compensation. Landowners play a crucial role on the development of a wind farm and some land parcels are more important to the success of the project than others. In order to advance the research on wind farm optimization, this paper relaxes the assumption that a continuous piece of land is available, developing a novel approach that includes landowners’ decisions on whether or not to participate in the project. The optimization results of this new approach show that, for a specific wind farm layout case, we can identify the most crucial landowners and the optimal positions of turbines prior to the negotiation process with landowners. Using this approach, a site developer can spend more resources on persuading these most-important landowners to take part in the project, or approach them in a personalized manner. This will ultimately increase the efficiency of wind farm projects, saving time and money in the development stages.

The change in electronic wavefunction brought about by optical excitation of a color center causes the surrounding ions to seek a new configuration of lower energy. Thus, optical excitation creates a momentary high level of localized vibrational excitation which the center must lose before it can emit in a Stokes shifted band. There are two schools of thought about how the vibrational relaxation takes place. One holds that the "configuration coordinate" (the most significant normal mode of ionic motion) must undergo a number of cycles of damped motion, shedding the excess energy in a sequence of optical phonons. The other [1] holds that the relaxation can take place in a fraction of the period of one optical phonon, and that the excess energy is released all at once in a shower of coherent phonons. Previous studies of impurity centers with only moderate coupling between electronic and ionic parts [2], or of a color center with a very large Stokes shift [3] seemed to indicate the former model, as the measured low temperature relaxation times were always many times the phonon period. However, we present here the first evidence that a color center (in this case the F2+, a single electron trapped by a pair of adjacent anion vacancies in an alkali halide crystal) relaxes according to the latter model. That is, we have made measurements indicating a relaxation time of less than one phonon period for that center.

The ability of adhesives to bond paper and paperboard is critical for most packaging and converting operations. Despite the huge body of literature describing both paper and adhesives technologies, there are only a few research papers describing paper/adhesive interactions. Described herein are the results of a systematic investigation of pressure sensitive adhesive (PSA) peeling from paper. The peel force versus peel distance curve depends upon the failure mode. A constant force is observed when the PSA cleanly separates from paper (i.e. interfacial failure) at low peel rate. By contrast, at high peeling rates, in the paper failure domain, the peel force climbs to a maximum and then relaxes to a steady-state value. The maximum peel force, which we call the peak force, corresponds to the fracture of the top layer of fibres during the initiation of paper delamination whereas the steady-state peel force occurs during the propagation of paper delamination. To characterize the range of behaviors it is necessary to conduct a series of peeling experiments over an extended range of peel rates. The results are best analyzed by plotting the peak peel force versus the peel rate on logarithmic axes giving what we call a peel map. For a broad range of tape/paper combinations, peel maps have similar shapes. The interfacial failure domain consists of a linear segment with a positive slope. This line intersects with a horizontal line segment at higher peel rates, corresponding to the paper failure domain. Principal component analysis, a multivariate statistical analysis, of a large set of peel maps was used to reveal the influence of paper properties on peeling. The peak peel forces in the paper failure domain correlated with standard paper properties linked to z-directional strength. The slopes of the peel maps in the interfacial domain were independent of paper properties but were sensitive to adhesive rheology. The absolute location of the interfacial segment of the peel map mainly was sensitive to the chemical composition of the paper surface and secondarily related to surface roughness. Water contact angles on paper were not good predictors of adhesion. Finally, we illustrate the utility of peak peel force in the paper failure domain as a measure of paper surface strength.