Training an Ising machine with equilibrium propagation.

Ising machines, which are hardware implementations of the Ising model of coupled spins, have been influential in the development of unsupervised learning algorithms at the origins of Artificial Intelligence (AI). However, their application to AI has been limited due to the complexities in matching supervised training methods with Ising machine physics, even though these methods are essential for achieving high accuracy. In this study, we demonstrate an efficient approach to train Ising machines in a supervised way through the Equilibrium Propagation algorithm, achieving comparable results to software-based implementations. We employ the quantum annealing procedure of the D-Wave Ising machine to train a fully-connected neural network on the MNIST dataset. Furthermore, we demonstrate that the machine's connectivity supports convolution operations, enabling the training of a compact convolutional network with minimal spins per neuron. Our findings establish Ising machines as a promising trainable hardware platform for AI, with the potential to enhance machine learning applications.

Multilayer spintronic neural networks with radiofrequency connections.

Spintronic nano-synapses and nano-neurons perform neural network operations with high accuracy thanks to their rich, reproducible and controllable magnetization dynamics. These dynamical nanodevices could transform artificial intelligence hardware, provided they implement state-of-the-art deep neural networks. However, there is today no scalable way to connect them in multilayers. Here we show that the flagship nano-components of spintronics, magnetic tunnel junctions, can be connected into multilayer neural networks where they implement both synapses and neurons thanks to their magnetization dynamics, and communicate by processing, transmitting and receiving radiofrequency signals. We build a hardware spintronic neural network composed of nine magnetic tunnel junctions connected in two layers, and show that it natively classifies nonlinearly separable radiofrequency inputs with an accuracy of 97.7%. Using physical simulations, we demonstrate that a large network of nanoscale junctions can achieve state-of-the-art identification of drones from their radiofrequency transmissions, without digitization and consuming only a few milliwatts, which constitutes a gain of several orders of magnitude in power consumption compared to currently used techniques. This study lays the foundation for deep, dynamical, spintronic neural networks.

Spatial priorities for freshwater biodiversity conservation in light of catchment protection and connectivity in Europe.

Freshwater ecosystems host disproportionately high numbers of species relative to their surface area yet are poorly protected globally. We used data on the distribution of 1631 species of aquatic plant, mollusc, odonate and fish in 18,816 river and lake catchments in Europe to establish spatial conservation priorities based on the occurrence of threatened, range-restricted and endemic species using the Marxan systematic conservation planning tool. We found that priorities were highest for rivers and ancient lakes in S Europe, large rivers and lakes in E and N Europe, smaller lakes in NW Europe and karst/limestone areas in the Balkans, S France and central Europe. The a priori inclusion of well-protected catchments resulted in geographically more balanced priorities and better coverage of threatened (critically endangered, endangered and vulnerable) species. The a priori exclusion of well-protected catchments showed that priority areas that need further conservation interventions are in S and E Europe. We developed three ways to evaluate the correspondence between conservation priority and current protection by assessing whether a cathment has more (or less) priority given its protection level relative to all other catchments. Each method found that priority relative to protection was high in S and E Europe and generally low in NW Europe. The inclusion of hydrological connectivity had little influence on these patterns but decreased the coverage of threatened species, indicating a trade-off between connectivity and conservation of threatened species. Our results suggest that catchments in S and E Europe need urgent conservation attention (protected areas, restoration, management, species protection) in the face of imminent threats such as river regulation, dam construction, hydropower development and climate change. Our study presents continental-scale conservation priorities for freshwater ecosystems in ecologically meaningful planning units and will thus be important in freshwater biodiversity conservation policy and practice, and water management in Europe.

Different life stage, different risks: Thermal performance across the life cycle of Salmo trutta and Salmo salar in the face of climate change.

Extending assessments of climate change-induced range shifts via correlative species distribution models by including species traits is crucial for conservation planning. However, comprehensive assessments of future distribution scenarios incorporating responses of biotic factors are poorly investigated. Therefore, the aim of our study was to extend the understanding about the combined usage of species traits data and species distribution models for different life stages and distribution scenarios. We combine global model predictions for the 2050s and thermal performances of Salmo trutta and Salmo salar under consideration of different life stages (adults, juveniles, eggs), timeframes (monthly, seasonally, yearly), and dispersal scenarios (no dispersal, free dispersal, restricted dispersal). We demonstrate that thermal performances of different life stages will either increase or decrease for certain time periods. Model predictions and thermal performances imply range declines and poleward shifts. Dispersal to suitable habitats will be an important factor mitigating warming effects; however, dams may block paths to areas linked to high performances. Our results emphasize enhanced inclusion of critical periods for species and proper dispersal solutions in conservation planning.

PKA and AKIP1 interact to mediate cAMP-driven COX-2 expression: A potentially pivotal interaction in preterm and term labour.

Previously, we showed that cAMP increased COX-2 expression in myometrial cells via MAPK. Here, we have extended these observations, using primary myometrial cell cultures to show that the cAMP agonist, forskolin, enhances IL-1ß-driven COX-2 expression. We then explored the role of A-kinase interacting protein (AKIP1), which modulates the effect of PKA on p65 activation. AKIP1 knockdown reversed the effect of forskolin, such that its addition inhibited IL-1ß-induced COX-2 mRNA expression and reduced the IL-1ß-induced increase in nuclear levels of p65 and c-jun. Forskolin alone and with IL-1ß increased IκBα mRNA expression suggesting that in the context of inflammation and in the presence of AKIP1, cAMP enhances p65 activation. AKIP1 knockdown reversed these changes. Interestingly, AKIP1 knockdown had minimal effect on the ability of forskolin to repress either basal OTR expression or IL-1ß-stimulated OTR mRNA expression. AKIP1 was up-regulated by IL-1ß, but not stretch and was repressed by cAMP. The mRNA expression of AKIP1 increased in early labour in tandem with an increase in COX-2 mRNA and protein. AKIP1 protein levels were also increased with inflammation and stretch-induced preterm labour. Our results identify a second important cAMP effector-switch occurring at term in human myometrium and suggest that a hitherto unrecognized interaction may exist between AKIP1, NFκB and AP-1. These data add to the proposition that cAMP acts as a key regulator of human myometrial contractility.

Climatic and socioeconomic effects on land cover changes across Europe: Does protected area designation matter?

Land cover change is a dynamic phenomenon driven by synergetic biophysical and socioeconomic effects. It involves massive transitions from natural to less natural habitats and thereby threatens ecosystems and the services they provide. To retain intact ecosystems and reduce land cover change to a minimum of natural transition processes, a dense network of protected areas has been established across Europe. However, even protected areas and in particular the zones around protected areas have been shown to undergo land cover changes. The aim of our study was to compare land cover changes in protected areas, non-protected areas, and 1 km buffer zones around protected areas and analyse their relationship to climatic and socioeconomic factors across Europe between 2000 and 2012 based on earth observation data. We investigated land cover flows describing major change processes: urbanisation, afforestation, deforestation, intensification of agriculture, extensification of agriculture, and formation of water bodies. Based on boosted regression trees, we modelled correlations between land cover flows and climatic and socioeconomic factors. The results show that land cover changes were most frequent in 1 km buffer zones around protected areas (3.0% of all buffer areas affected). Overall, land cover changes within protected areas were less frequent than outside, although they still amounted to 18,800 km2 (1.5% of all protected areas) from 2000 to 2012. In some parts of Europe, urbanisation and intensification of agriculture still accounted for up to 25% of land cover changes within protected areas. Modelling revealed meaningful relationships between land cover changes and a combination of influencing factors. Demographic factors (accessibility to cities and population density) were most important for coarse-scale patterns of land cover changes, whereas fine-scale patterns were most related to longitude (representing the general east/west economic gradient) and latitude (representing the north/south climatic gradient).

Aminophylline and progesterone prevent inflammation-induced preterm parturition in the mouse†.

Although progesterone (P4) supplementation is the most widely used therapy for the prevention of preterm labor (PTL), reports of its clinical efficacy have been conflicting. We have previously shown that the anti-inflammatory effects of P4 can be enhanced by increasing intracellular cyclic adenosine monophosphate (cAMP) levels in primary human myometrial cells. Here, we have examined whether adding aminophylline (Am), a non-specific phosphodiesterase inhibitor that increases intracellular cAMP levels, to P4 might improve its efficacy using in vivo and in vitro models of PTL. In a mouse model of lipopolysaccharide (LPS)-induced PTL, we found that the combination of P4 and Am delayed the onset of LPS-induced PTL, while the same dose of P4 and Am alone had no effect. Pup survival was not improved by either agent alone or in combination. Myometrial prolabor and inflammatory cytokine gene expression was reduced, but the reduction was similar in P4 and P4/Am treated mice. There was no effect of the combination of P4 and Am on an ex vivo assessment of myometrial contractility. In human myometrial cells and myometrial tissue explants, we found that the combination had marked anti-inflammatory effects, reducing cytokine and COX-2 mRNA and protein levels to a greater extent than either agent alone. These data suggest that the combination of P4 and Am has a more potent anti-inflammatory effect than either agent alone and may be an effective combination in women at high-risk of PTL.

Freshwater species distributions along thermal gradients.

The distribution of a species along a thermal gradient is commonly approximated by a unimodal response curve, with a characteristic single optimum near the temperature where a species is most likely to be found, and a decreasing probability of occurrence away from the optimum. We aimed at identifying thermal response curves (TRCs) of European freshwater species and evaluating the potential impact of climate warming across species, taxonomic groups, and latitude. We first applied generalized additive models using catchment-scale global data on distribution ranges of 577 freshwater species native to Europe and four different temperature variables (the current annual mean air/water temperature and the maximum air/water temperature of the warmest month) to describe species TRCs. We then classified TRCs into one of eight curve types and identified spatial patterns in thermal responses. Finally, we integrated empirical TRCs and the projected geographic distribution of climate warming to evaluate the effect of rising temperatures on species' distributions. For the different temperature variables, 390-463 of 577 species (67.6%-80.2%) were characterized by a unimodal TRC. The number of species with a unimodal TRC decreased from central toward northern and southern Europe. Warming tolerance (WT = maximum temperature of occurrence-preferred temperature) was higher at higher latitudes. Preferred temperature of many species is already exceeded. Rising temperatures will affect most Mediterranean species. We demonstrated that freshwater species' occurrence probabilities are most frequently unimodal. The impact of the global climate warming on species distributions is species and latitude dependent. Among the studied taxonomic groups, rising temperatures will be most detrimental to fish. Our findings support the efforts of catchment-based freshwater management and conservation in the face of global warming.

Progesterone, the maternal immune system and the onset of parturition in the mouse.

The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2), and the onset of parturition was examined in (i) naïve mice delivering at term; (ii) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and (iii) in mice treated with P4 to prevent term parturition. In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43, and COX-2 increased with, but not before, parturition. With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9 h post-RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition. In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented, and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased. These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabor factor synthesis via mRNA-dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation.

Vulnerability of European freshwater catchments to climate change.

Climate change is expected to exacerbate the current threats to freshwater ecosystems, yet multifaceted studies on the potential impacts of climate change on freshwater biodiversity at scales that inform management planning are lacking. The aim of this study was to fill this void through the development of a novel framework for assessing climate change vulnerability tailored to freshwater ecosystems. The three dimensions of climate change vulnerability are as follows: (i) exposure to climate change, (ii) sensitivity to altered environmental conditions and (iii) resilience potential. Our vulnerability framework includes 1685 freshwater species of plants, fishes, molluscs, odonates, amphibians, crayfish and turtles alongside key features within and between catchments, such as topography and connectivity. Several methodologies were used to combine these dimensions across a variety of future climate change models and scenarios. The resulting indices were overlaid to assess the vulnerability of European freshwater ecosystems at the catchment scale (18 783 catchments). The Balkan Lakes Ohrid and Prespa and Mediterranean islands emerge as most vulnerable to climate change. For the 2030s, we showed a consensus among the applied methods whereby up to 573 lake and river catchments are highly vulnerable to climate change. The anthropogenic disruption of hydrological habitat connectivity by dams is the major factor reducing climate change resilience. A gap analysis demonstrated that the current European protected area network covers <25% of the most vulnerable catchments. Practical steps need to be taken to ensure the persistence of freshwater biodiversity under climate change. Priority should be placed on enhancing stakeholder cooperation at the major basin scale towards preventing further degradation of freshwater ecosystems and maintaining connectivity among catchments. The catchments identified as most vulnerable to climate change provide preliminary targets for development of climate change conservation management and mitigation strategies.

Estrogen alters the splicing of type 1 corticotropin-releasing hormone receptor in breast cancer cells.

Hormonal stress response is associated with the pathogenesis of disease, including cancer. The role of the stress hormone CRH (corticotropin-releasing hormone) in breast cancer is complex, and its abundance and biological activity may be modulated by estrogen. In the estrogen receptor-positive (ER+) malignant mammary epithelial cell line MCF7, CRH activated numerous kinases and downstream effectors, at least some of which were mediated by the CRH receptor type 1 (CRH-R1). CRH also increased the transcription of many genes that encode effectors, transcriptional targets, or regulators associated with estrogen signaling. Estrogen increased the abundance of the mRNA encoding CRH-R2 and an alternative splice variant encoding CRH-R1 in which exon 12 was deleted [CRH-R1(Δ12)]. Estrogen inhibited the expression SRSF6, which encodes serine/arginine-rich splicing factor 55 (SRp55). An increase in CRH-R1(Δ12), in response to either estrogen or SRp55 knockdown, dampened the cellular response to CRH and prevented its inhibitory effects on cell invasion. SRp55 knockdown also induced additional splicing events within exons 9 to 12 of CRH-R1, whereas overexpression of SRp55 prevented estrogen-induced generation of CRH-R1(Δ12). ER+ breast tumors had increased CRH-R2 and CRH-R1(Δ12) mRNA abundance, which was associated with decreased abundance of the mRNA encoding SRp55, compared with the amounts in ER- tumors, suggesting that estrogen contributes to the pathophysiology of ER+ breast cancer by altering CRH receptor diversity and disrupting CRH-mediated signaling.

Corticotropin-releasing hormone interacts with interleukin-1ß to regulate prostaglandin H synthase-2 expression in human myometrium during pregnancy and labor.

CONTEXT: The onset of labor appears to involve the activation of myometrial inflammatory pathways, and transcription factors such as nuclear factor-κB (NF-κB) control expression of the contraction-associated proteins required to induce a procontractile phenotype. These responses might involve CRH, which integrates immune and neuroendocrine systems. OBJECTIVES: In human myometrium we investigated cyclooxygenase 2 (PGHS2) expression and regulation by CRH and the proinflammatory cytokine IL-1ß before and after labor. DESIGN: Myometrial tissues obtained from pregnant women at term before (n = 12) or during labor (n = 10) and pathological cases of choriamnionitis-associated term labor (n = 5) were used to isolate primary myocytes and investigate in vitro, CRH effects on basal and IL-1ß regulated p65 activation and PGHS2 expression. RESULTS: In nonlaboring myometrial cells, CRH was unable to induce NF-κB nuclear translocation; however, it altered the temporal dynamics of IL-1ß-driven NF-κB nuclear entry by initially delaying entry and subsequently prolonging retention. These CRH-R1-driven effects were associated with a modest inhibitory action in the early phase (within 2 hours) of IL-1ß stimulated PGHS2 mRNA expression, whereas prolonged stimulation for 6-18 hours augmented the IL-1ß effects. The early-phase effect required intact protein kinase A activity and was diminished after the onset of labor. The presence of chorioamnionitis led to exaggerated PGHS2 mRNA responses to IL-1ß but diminished effects of CRH. CONCLUSIONS: CRH is involved in the inflammatory regulation of PGHS2 expression before and during labor; these actions might be important in priming and preparing the myometrium for labor and cellular adaptive responses to inflammatory mediators.

Alternative mRNA splicing of G protein-coupled receptors.

Alternative mRNA splicing as an important mechanism for generating protein diversity has been implicated to affect 60-70% of human genes (Wang et al., 2008). The G protein-coupled receptors (GPCRs), the largest group of membrane receptors, have a broad distribution and function. Posttranslational modifications or/and association with regulatory proteins can govern cell- and tissue-specific pharmacological, signaling, and regulatory characteristics of GPCRs. However, increasing body of evidence suggests that alternative splicing of GPCRs is a mechanism that can modulate GPCRs' function (Einstein et al., 2008). Many GPCRs have a potential to undergo alternative pre-mRNA splicing (~50%). Due to a general lack of isoform-specific antibodies, GPCRs' alternative splicing is mainly studied on mRNA level employing various PCR techniques. Functional characteristics (including signaling and structure) of alternatively spliced GPCRs are studied in overexpression system employing standard biochemical techniques. In this chapter, we will describe the methods for detection and quantification of alternatively spliced GPCR mRNA. As the experimental paradigm, we use type 1 corticotropin-releasing hormone receptor (CRH-R1).

Where are all the fish: potential of biogeographical maps to project current and future distribution patterns of freshwater species.

The dendritic structure of river networks is commonly argued against use of species atlas data for modeling freshwater species distributions, but little has been done to test the potential of grid-based data in predictive species mapping. Using four different niche-based models and three different climate change projections for the middle of the 21st century merged pairwise as well as within a consensus modeling framework, we studied the variability in current and future distribution patterns of 38 freshwater fish species across Germany. We used grid-based (11×11 km) fish distribution maps and numerous climatic, topographic, hydromorphologic, and anthropogenic factors derived from environmental maps at a finer scale resolution (250 m-1 km). Apart from the explicit predictor selection, our modeling framework included uncertainty estimation for all phases of the modeling process. We found that the predictive performance of some niche-based models is excellent independent of the predictor data set used, emphasizing the importance of a well-grounded predictor selection process. Though important, climate was not a primary key factor for any of the studied fish species groups, in contrast to substrate preferences, hierarchical river structure, and topography. Generally, distribution ranges of cold-water and warm-water species are expected to change significantly in the future; however, the extent of changes is highly uncertain. Finally, we show that the mismatch between the current and future ranges of climatic variables of more than 90% is the most limiting factor regarding reliability of our future estimates. Our study highlighted the underestimated potential of grid cell information in biogeographical modeling of freshwater species and provides a comprehensive modeling framework for predictive mapping of species distributions and evaluation of the associated uncertainties.

FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists.

BACKGROUND AND OBJECTIVE: Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET). METHODS: Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M(1) mAChR, respectively. The optimized FRET receptor construct (M(1)-cam5) was expressed stably in HEK293 cells. RESULTS: The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gα(q/11) proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M(1) FRET (F(EYFP)/F(ECFP)) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine. CONCLUSION: The M(1) FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M(1) mAChR activation.

Molecular determinants and feedback circuits regulating type 2 CRH receptor signal integration.

In most target tissues, the adenylyl cyclase/cAMP/PKA, the extracellular signal regulated kinase and the protein kinase B/Akt are the main pathways employed by the type 2 corticotropin-releasing hormone receptor to mediate the biological actions of urocortins (Ucns) and CRH. To decipher the molecular determinants of CRH-R2 signaling, we studied the signaling pathways in HEK293 cells overexpressing recombinant human CRH-R2ß receptors. Use of specific kinase inhibitors showed that the CRH-R2ß cognate agonist, Ucn 2, activated extracellular signal regulated kinase in a phosphoinositide 3-kinase and cyclic adenosine monophosphate/PKA-dependent manner with contribution from Epac activation. Ucn 2 also induced PKA-dependent association between AKAP250 and CRH-R2ß that appeared to be necessary for extracellular signal regulated kinase activation. PKB/Akt activation was also mediated via pertussis toxin-sensitive G-proteins and PI3-K activation but did not require cAMP/PKA, Epac or protein kinase C for optimal activation. Potential feedback mechanisms that target the CRH-R2ß itself and modulate receptor trafficking and endocytosis were also investigated. Indeed, our results suggested that inhibition of either PKA or extracellular signal regulated kinase pathway accelerates CRH-R2ß endocytosis. Furthermore, Ucn 2-activated extracellular signal regulated kinase appeared to target ß-arrestin1 and modulate, through phosphorylation at Ser412, ß-arrestin1 translocation to the plasma membrane and CRH-R2ß internalization kinetics. Loss of this "negative feedback" mechanism through inhibition of the extracellular signal regulated kinase activity resulted in significant attenuation of Ucn 2-induced cAMP response, whereas Akt phosphorylation was not affected by altered receptor endocytosis. These findings reveal a complex interplay between the signaling molecules that allow "fine-tuning" of CRH-R2ß functional responses and regulate signal integration. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Characterization of structural determinants of type 1 corticotropin releasing hormone (CRH) receptor signalling properties.

Mammalian adaptation to stressful stimuli requires activation of the type 1 corticotropin releasing hormone (CRH) receptor (CRH-R1), a 415 amino acid protein that belongs to the large superfamily of 7 transmembrane domain receptors that relay signals across cells through activation of G-proteins. CRH-R1 expression and activity is regulated at the gene level by mRNA alternative splicing that results in a number of CRH-R1 variants. This process can generate putative CRH-R1 receptor variants with distinct structural and signaling properties; their study can provide important insights about the structural determinants of CRH-R1 functional characteristics. Using site-directed mutagenesis by overlap extension polymerase chain reaction (OE-PCR), we investigated the structure-function relationships of a CRH-R1 variant with an extended 1st intracellular loop (IC1) (CRH-R1beta), a sequence modification that impairs signaling activity (such as cAMP production and MAPK activation). We identified a penta-amino acid cassette within the 29-amino acid insert of CRH-R1beta rich in positive charged amino acids (F(170)-R(174)), as an important structural determinant for the impaired cAMP response.

Identification of a novel corticotropin-releasing hormone type 1beta-like receptor variant lacking Exon 13 in human pregnant myometrium regulated by estradiol-17beta and progesterone.

Two types of CRH receptors mediate the diverse biological functions of CRH and CRH-related peptides. The type 1 CRH-R (CRH-R1) is extensively targeted by pre-mRNA splicing mechanisms that give rise to multiple mRNA splice variants. RT-PCR amplification of CRH-R1 sequences from human myometrium yielded cDNAs that encode a novel CRH-R1 splice variant with structural characteristics identical with CRH-R1ß except a 14-amino acid deletion in the seventh transmembrane domain characteristic of the CRH-R1d. Transient expression of the hybrid CRH-R1 variant (CRH-R1ß/d) in human embryonic kidney 293 cells revealed primarily intracellular expression, although some plasma membrane protein expression was also detectable. CRH bound to CRH-R1ß/d with affinity comparable with the CRH-R1ß; however, it was unable to stimulate adenylyl cyclase or other second messengers. Using a semiquantitative RT-PCR assay, CRH-R1ß/d mRNA transcript was detected in human pregnant, but not nonpregnant, myometrium as early as 31 wk of gestation. Furthermore, in human pregnant myometrial cells, the relative expression of CRH-R1ß and CRH-R1ß/d mRNA appeared to be regulated by steroids; CRH-R1ß/d mRNA expression was increased by estradiol-17ß, whereas CRH-R1ß mRNA levels were increased by progesterone. Progesterone also substantially increased CRH-R1α mRNA levels and cellular responsiveness to CRH as determined by increased agonist binding and cAMP production as well as resistance to CRH-R heterologous desensitization by phorbol esters. These results provide novel evidence for distinct patterns of CRH-R1 splicing and identify specific steroid-mediated regulation of CRH-R1 variant expression, which might be important for modulating CRH actions during human pregnancy and labour.

Focus on the splicing of secretin GPCRs transmembrane-domain 7.

The family of G-protein coupled receptors (GPCRs) is one of the largest protein families in the mammalian genome with a fundamental role in cell biology. GPCR activity is finely tuned by various transcriptional, post-transcriptional and post-translational mechanisms. Alternative pre-mRNA splicing is now emerging as a crucial process regulating GPCR biological function. Intriguingly, this mechanism appears to extensively target the Secretin family of GPCRs, especially the exon that encodes a 14 amino acid sequence that forms the distal part of 7th transmembrane helix, and exhibits an unusually high level of sequence conservation among most Secretin GPCRs. Do the "TMD7-short" receptor variants have a role as novel regulators of GPCR signallng and, if so, what are the implications for hormonal actions and physiology?