Non-Immunosuppressive Cyclophilin Inhibitors.

Cyclophilins, enzymes with peptidyl-prolyl cis/trans isomerase activity, are relevant to a large variety of biological processes. The most abundant member of this enzyme family, cyclophilin A, is the cellular receptor of the immunosuppressive drug cyclosporine A (CsA). As a consequence of the pathophysiological role of cyclophilins, particularly in viral infections, there is a broad interest in cyclophilin inhibition devoid of immunosuppressive activity. This Review first gives an introduction into the physiological and pathophysiological roles of cyclophilins. The presentation of non-immunosuppressive cyclophilin inhibitors will commence with drugs based on chemical modifications of CsA. The naturally occurring macrocyclic sanglifehrins have become other lead structures for cyclophilin-inhibiting drugs. Finally, de novo designed compounds, whose structures are not derived from or inspired by natural products, will be presented. Relevant synthetic concepts will be discussed, but the focus will also be on biochemical studies, structure-activity relationships, and clinical studies.

Evolution of crassulacean acid metabolism (CAM) as an escape from ecological niche conservatism in Malagasy Bulbophyllum (Orchidaceae).

Despite growing evidence that niche shifts are more common in flowering plants than previously thought, little is known of whether such shifts are promoted by changes in photosynthetic pathways. Here we combine the most complete phylogeny for epiphytic Malagasy Bulbophyllum orchids (c. 210 spp.) with climatic niche and carbon isotope ratios to infer the group's spatial-temporal history, and the role of strongly expressed crassulacean acid metabolism (CAM) in facilitating niche shifts and diversification. We find that most extant species still retain niche (Central Highland) and photosynthesis (C3 ) states as present in the single mid-Miocene (c. 12.70 million yr ago (Ma)) ancestor colonizing Madagascar. However, we also infer a major transition to CAM, linked to a late Miocene (c. 7.36 Ma) invasion of species from the sub-humid highland first into the island's humid eastern coastal, and then into the seasonally dry 'Northwest Sambirano' rainforests, yet without significant effect on diversification rates. These findings indicate that CAM in tropical epiphytes may be selectively advantageous even in high rainfall habitats, rather than presenting a mere adaptation to dry environments or epiphytism per se. Overall, our study qualifies CAM as an evolutionary 'gateway' trait that considerably widened the spatial-ecological amplitude of Madagascar's most species-rich orchid genus.

Molecular phylogenetics and floral evolution of the Cirrhopetalum alliance (Bulbophyllum, Orchidaceae): Evolutionary transitions and phylogenetic signal variation.

The Cirrhopetalum alliance is a loosely circumscribed species-rich group within the mega-diverse genus Bulbophyllum (Orchidaceae). The monophyletic status of the alliance has been challenged by previous studies, although established sectional classifications have yet to be tested in a phylogenetic context. We used maximum likelihood and Bayesian analyses of DNA sequence data (cpDNA: matK and psbA-trnH; nrDNA: ITS and Xdh; 3509 aligned characters; 117 taxa), including all sections putatively associated with the Cirrhopetalum alliance, to reconstruct the phylogeny. We mapped 11 selected categorical floral characters onto the phylogeny to identify synapomorphies and assess potential evolutionary transitions across major clades. Our results unequivocally support the recognition of an amended Cirrhopetalum alliance as a well-supported monophyletic group characterized by clear synapomorphies, following the inclusion of sect. Desmosanthes and the exclusion of five putative Cirrhopetalum-allied sections. Most sections within the Cirrhopetalum alliance are demonstrated to be polyphyletic or paraphyletic, necessitating a new sectional classification. The inclusion of sect. Desmosanthes revolutionizes our understanding of the alliance, with significant evolutionary transitions in floral characters detected. We further investigated six continuously variable characters of the sepals and labellum, and detect phylogenetic conservatism in labellum width and the evolutionary lability of lateral sepal length, which can partly be explained by the different functional roles they play in pollination and pollinator trapping.

Wind pollination over 70 years reduces the negative genetic effects of severe forest fragmentation in the tropical oak Quercus bambusifolia.

Whether wind pollination in trees can offset the negative genetic consequences of anthropogenic forest fragmentation is not clearly established. To answer this question, we examined the demographic genetics of Quercus bambusifolia over a 70-year recovery period in highly fragmented forests in Hong Kong. We sampled 1138 individuals from 37 locations, and genetically analysed the chronosequence through the classification of tree diameters from the same populations using 13 microsatellite markers. Our study reveals that severe fragmentation caused a significant genetic bottleneck with very few remaining but genetically diverse individuals. We observed an enhanced genetic diversity during demographic recovery. We found full-sibs within populations and half-sibs across the study range. This reflects a limited seed dispersal and extensive pollen flow. Despite reduced genetic structure both among and within populations, overall a strong persisting genetic differentiation (F'ST = 0.240, P < 0.01) and significant small-scale spatial genetic structure (F(1) = 0.13, Sp = 0.024, P < 0.01) were observed. Existing bottlenecks and low effective population sizes within the temporal chronosequence suggest that the long-term effect of severe fragmentation cannot be entirely eliminated by wind pollination with demographic recovery in the absence of effective seed dispersal. Our results lead to recommendations for forest management.

Direct and indirect effects of climate on richness drive the latitudinal diversity gradient in forest trees.

Climate is widely recognised as an important determinant of the latitudinal diversity gradient. However, most existing studies make no distinction between direct and indirect effects of climate, which substantially hinders our understanding of how climate constrains biodiversity globally. Using data from 35 large forest plots, we test hypothesised relationships amongst climate, topography, forest structural attributes (stem abundance, tree size variation and stand basal area) and tree species richness to better understand drivers of latitudinal tree diversity patterns. Climate influences tree richness both directly, with more species in warm, moist, aseasonal climates and indirectly, with more species at higher stem abundance. These results imply direct limitation of species diversity by climatic stress and more rapid (co-)evolution and narrower niche partitioning in warm climates. They also support the idea that increased numbers of individuals associated with high primary productivity are partitioned to support a greater number of species.

Pollen-mediated gene flow ensures connectivity among spatially discrete sub-populations of Phalaenopsis pulcherrima, a tropical food-deceptive orchid.

BACKGROUND: Gene flow in plants via pollen and seeds is asymmetrical at different geographic scales. Orchid seeds are adapted to long-distance wind dispersal but pollinium transfer is often influenced by pollinator behavior. We combined field studies with an analysis of genetic diversity among 155 physically mapped adults and 1105 F1 seedlings to evaluate the relative contribution of pollen and seed dispersal to overall gene flow among three sub-populations of the food-deceptive orchid Phalaenopsis pulcherrima on Hainan Island, China. RESULTS: Phalaenopsis pulcherrima is self-sterile and predominantly outcrossing, resulting in high population-level genetic diversity, but plants are clumped and exhibit fine-scale genetic structuring. Even so, we detected low differentiation among sub-populations, with polynomial regression analysis suggesting gene flow via seed to be more restricted than that via pollen. Paternity analysis confirmed capsules of P. pulcherrima to each be sired by a single pollen donor, probably in part facilitated by post-pollination stigma obfuscation, with a mean pollen flow distance of 272.7 m. Despite limited sampling, we detected no loss of genetic diversity from one generation to the next. CONCLUSIONS: Outcrossing mediated by deceptive pollination and self-sterility promote high genetic diversity in P. pulcherrima. Long-range pollinia transfer ensures connectivity among sub-populations, offsetting the risk of genetic erosion at local scales.

Conservation impacts of commercial cultivation of endangered and overharvested plants.

Overharvesting is one of the greatest threats to species survival. Farming overharvested species is a conservation strategy that can meet growing market demand and conserve wild populations of the target species. This strategy is compatible with the international community's desire to uphold the right of local communities to use biological resources to support their livelihoods. However, studies investigating whether farming can alleviate poaching pressure have focused almost exclusively on animals. To address the shortfall in plant-focused studies, we compiled information on commercial cultivation of threatened plants to assess its conservation benefits. Because China's rising middle class has rapidly intensified demand for wildlife products, we searched the scientific literature published in Chinese (China National Knowledge Infrastructure and Baidu) and in English. We found 32 reports that contained data on 193 internationally or nationally threatened plant species that were under commercial cultivation. These reports showed that cultivations of 82% of the 193 species were sustained by collecting whole plants from the wild periodically or continuously. Although based on a small sample size, species that were maintained in cultivation only through artificial propagation or seeds collected in the wild were likely associated with a reported reduction in wild harvesting of whole plants. Even so, results of correlation analyses suggested that production system, scale, and when a species began being cultivated had little effect on conservation status of the species, either globally or in China. However, species brought into cultivation relatively recently and on a smaller scale were more likely to have undergone a reduction in collecting pressure. Farming of nonmedicinal plants was most problematic for species conservation because wild plants were laundered (i.e., sold as cultivated plants). For effective conservation, policy to guide cultivation operations based on the target species' biological characteristics, cultural significance, market demand, and conservation status is needed.

Impactos en la Conservación del Cultivo Comercial de Plantas Sobreexplotadas y en Peligro de Extinción Resumen La sobreexplotación es una de las mayores amenazas para la supervivencia de una especie. El cultivo de especies sobreexplotadas es una estrategia de conservación que puede cumplir con la demanda creciente en el mercado y a la vez conservar especies silvestres de la especie diana. Esta estrategia es compatible con el deseo de la comunidad internacional de defender el derecho que tienen las comunidades locales a usar los recursos biológicos para mantener su sustento. Sin embargo, los estudios que indagan si el cultivo puede aliviar la presión de la colecta furtiva se han enfocado casi exclusivamente en animales. Para tratar con este déficit de estudios enfocados en plantas compilamos información sobre el cultivo comercial de plantas amenazadas para evaluar los beneficios de conservación del cultivo comercial. Ya que la creciente clase media china ha intensificado rápidamente la demanda de productos silvestres decidimos buscar en la literatura científica en chino (Infraestructura Nacional de Conocimiento de China y Baidu) y en inglés. Encontramos 32 reportes que contenían datos sobre 193 especies de plantas amenazadas nacional o internacionalmente que se encontraban en cultivos comerciales. Estos reportes mostraron que los cultivos del 82% de las 193 especies están sostenidos por la colecta de plantas completas en el campo de manera periódica o continua. Aunque nos basamos en un pequeño tamaño de muestra, las especies que se mantenían en cultivos sólo mediante la propagación artificial o mediante semillas recolectadas en campo tenían probabilidad de estar asociadas con una reducción reportada de la cosecha silvestre de plantas completas. Aún así, los resultados de los análisis de correlación sugieren que el sistema de producción, la escala, y cuándo se comenzó a cultivar a las especies tuvieron el menor efecto sobre el estado de conservación de la especie, fuera a escala mundial o en China. Sin embargo, las especies que se han introducido recientemente al cultivo y a menor escala tenían mayor probabilidad de haber sufrido una reducción en la presión de colecta. El cultivo de plantas no medicinales fue la más problemática para la conservación de especies ya que las plantas silvestres eran "lavadas" (es decir, vendidas como plantas cultivadas). Para una conservación efectiva se necesita de políticas que guíen las operaciones de cultivo con base en las características biológicas, la importancia cultural, la demanda en el mercado y el estado de conservación de la especie de interés.

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays.

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP-dependent kinase, inhibition of mTOR signaling, and induction of cell-cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.

Targeting Extracellular Cyclophilin A Reduces Neuroinflammation and Extends Survival in a Mouse Model of Amyotrophic Lateral Sclerosis.

Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1G93A mice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1G93A mouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients.SIGNIFICANCE STATEMENT We provide evidence that extracellular cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), is a mediator of the neuroinflammatory reaction in amyotrophic lateral sclerosis (ALS) and is toxic for motor neurons. Supporting this, a specific extracellular PPIA inhibitor reduced neuroinflammation, rescued motor neurons, and extended survival in the SOD1G93A mouse model of familial ALS. Our findings suggest selective pharmacological inhibition of extracellular PPIA as a novel therapeutic strategy, not only for SOD1-linked ALS, but possibly also for sporadic ALS. This approach aims to address the neuroinflammatory reaction that is a major hallmark of ALS. However, given the complexity of the disease, a combination of therapeutic approaches may be necessary.

A chemical compound inhibiting the Aha1-Hsp90 chaperone complex.

The eukaryotic Hsp90 chaperone machinery comprises many co-chaperones and regulates the conformation of hundreds of cytosolic client proteins. Therefore, it is not surprising that the Hsp90 machinery has become an attractive therapeutic target for diseases such as cancer. The compounds used so far to target this machinery affect the entire Hsp90 system. However, it would be desirable to achieve a more selective targeting of Hsp90-co-chaperone complexes. To test this concept, in this-proof-of-principle study, we screened for modulators of the interaction between Hsp90 and its co-chaperone Aha1, which accelerates the ATPase activity of Hsp90. A FRET-based assay that monitored Aha1 binding to Hsp90 enabled identification of several chemical compounds modulating the effect of Aha1 on Hsp90 activity. We found that one of these inhibitors can abrogate the Aha1-induced ATPase stimulation of Hsp90 without significantly affecting Hsp90 ATPase activity in the absence of Aha1. NMR spectroscopy revealed that this inhibitory compound binds the N-terminal domain of Hsp90 close to its ATP-binding site and overlapping with a transient Aha1-interaction site. We also noted that this inhibitor does not dissociate the Aha1-Hsp90 complex but prevents the specific interaction with the N-terminal domain of Hsp90 required for catalysis. In consequence, the inhibitor affected the activation and processing of Hsp90-Aha1-dependent client proteins in vivo We conclude that it is possible to abrogate a specific co-chaperone function of Hsp90 without inhibiting the entire Hsp90 machinery. This concept may also hold true for other co-chaperones of Hsp90.

Screening for Selective Protein Inhibitors by Using the IANUS Peptide Array.

Finding new road blacks: A peptidic inhibitor of calcineurin (CaN)-mediated nuclear factor of activated T cells (NFAT) dephosphorylation, which is developed through a template-assisted IANUS (Induced orgANisation of strUcture by matrix-assisted togethernesS) peptide array, is cell permeable and able to block the translocation of green fluorescent protein-NFAT fusion protein (GFP-NFAT) into the nucleus after stimulation.

Synthesis and biochemical evaluation of two novel N-hydroxyalkylated cyclosporin A analogs.

The cyclic undecapeptide cyclosporin A (CsA) is a widely used immunosuppressive agent. Its immunosuppressive properties arise from strong binding to cyclophilins (Cyp) followed by inhibition of the protein calcineurin (CaN) by the binary CsA/Cyp complex and subsequent negative regulation of T-cell activation. In the present study we show a novel way to modify the CsA ring by selective N-hydroxyalkylation of the residues Val5 and d-Ala8. Moreover, the influence of these structural CsA modifications on the ability of the CsA analogs to bind Cyp, to inhibit CaN and to penetrate membranes of living cells was investigated. Our results show that the Val5 N-substitution significantly improved compound cell-permeability and markedly diminished CaN inhibition by the binary CsA analog/CypA complex but to a lesser extent Cyp inhibition. In contrast, the N-alkylation of d-Ala8 gave a product with significantly reduced affinity for Cyp but its immunosuppressive effects remained similar to CsA. Possible explanations of the observed experimental data are provided by computational studies.

Species composition of the international shark fin trade assessed through a retail-market survey in Hong Kong.

The shark fin trade is a major driver of shark exploitation in fisheries all over the world, most of which are not managed on a species-specific basis. Species-specific trade information highlights taxa of particular concern and can be used to assess the efficacy of management measures and anticipate emerging threats. The species composition of the Hong Kong Special Administrative Region of China, one of the world's largest fin trading hubs, was partially assessed in 1999-2001. We randomly selected and genetically identified fin trimmings (n = 4800), produced during fin processing, from the retail market of Hong Kong in 2014-2015 to assess contemporary species composition of the fin trade. We used nonparametric species estimators to determine that at least 76 species of sharks, batoids, and chimaeras supplied the fin trade and a Bayesian model to determine their relative proportion in the market. The diversity of traded species suggests species substitution could mask depletion of vulnerable species; one-third of identified species are threatened with extinction. The Bayesian model suggested that 8 species each comprised >1% of the fin trimmings (34.1-64.2% for blue [Prionace glauca], 0.2-1.2% for bull [Carcharhinus leucas] and shortfin mako [Isurus oxyrinchus]); thus, trade was skewed to a few globally distributed species. Several other coastal sharks, batoids, and chimaeras are in the trade but poorly managed. Fewer than 10 of the species we modeled have sustainably managed fisheries anywhere in their range, and the most common species in trade, the blue shark, was not among them. Our study and approach serve as a baseline to track changes in composition of species in the fin trade over time to better understand patterns of exploitation and assess the effects of emerging management actions for these animals.

Preponderance of clonality triggers loss of sex in Bulbophyllum bicolor, an obligately outcrossing epiphytic orchid.

Vegetative propagation (clonal growth) conveys several evolutionary advantages that positively affect life history fitness and is a widespread phenomenon among angiosperms that also reproduce sexually. However, a bias towards clonality can interfere with sexual reproduction and lead to sexual extinction, although a dearth of effective genetic tools and mathematical models for clonal plants has hampered assessment of these impacts. Using the endangered tropical epiphytic or lithophytic orchid Bulbophyllum bicolor as a model, we integrated an examination of breeding system with 12 microsatellite loci and models valid for clonal species to test for the "loss of sex" and infer likely consequences for long-term reproductive dynamics. Bagging experiments and field observations revealed B. bicolor to be self-incompatible and pollinator-dependent, with an absence of fruit-set over 4 years. Challenging the assumptions that clonal populations can be as genotypically diverse as sexually reproducing ones and that clonality does not greatly influence genetic structure, just 22 multilocus genotypes were confirmed among all 15 extant natural populations, 12 of the populations were found to be monoclonal, and all three multiclonal ones exhibited a distinct phalanx clonal architecture. Our results suggest that all B. bicolor populations depend overwhelmingly on clonal growth for persistence, with a concomitant loss of sex due to an absence of pollinators and a lack of mating opportunities at virtually all sites, both of which are further entrenched by habitat fragmentation. Such cryptic life history impacts, potentially contributing to extinction debt, could be widespread among similarly fragmented, outcrossing tropical epiphytes, demanding urgent conservation attention.

Identification of low abundance cyclophilins in human plasma.

Cylophilins (Cyps) belong to the ubiquitously distributed enzyme class of peptidyl prolyl cis/trans isomerases (EC5.2.1.8), which are foldases capable of accelerating slow steps in the refolding of denatured proteins. At least 20 different Cyp isoenzymes are broadly distributed among all organs and cellular compartments in humans. Extracellularly localized Cyps came into the scientific focus recently because of their involvement in the control of inflammatory diseases, as well as viral and bacterial infections. However, detailed insights into Cyp functions are often hampered by the lack of sensitive detection methods. We present an improved method for affinity purification and detection of Cyp in biotic samples in this manuscript. The procedure takes advantage of two novel cyclosporine A derivatives. Derivative 1 was used to capture Cyps from the sample while derivative 2 was applied for selective release from the affinity matrix. Using this approach, eight different Cyp (CypA, CypB, CypC, Cyp40 (PPID), CypE, CypD (PPIF), CypH, and CypL1) were unambiguously detected in healthy human blood plasma. Moreover, extracellular CypA was found to be partially modified by Nε acetylation on residues Lys44, Lys133, Lys155, as well as Nα  acetylation at the N-terminal Val residue. Nα  acetylation of Ser2 residue was also found for Cyp40.

Frequent but asymmetric niche shifts in Bulbophyllum orchids support environmental and climatic instability in Madagascar over Quaternary time scales.

BACKGROUND: Species or clades may retain or shift their environmental niche space over evolutionary time. Understanding these processes offers insights into the environmental processes fuelling lineage diversification and might also provide information on past range dynamics of ecosystems. However, little is known about the relative contributions of niche conservatism versus niche divergence to species diversification in the tropics. Here, we examined broad-scale patterns of niche evolution within a Pliocene-Pleistocene clade of epiphytic Bulbophyllum orchids (30 spp.) whose collective distribution covers the northwest and eastern forest ecosystems of Madagascar. RESULTS: Using species occurrence data, ecological niche models, and multivariate analyses of contributing variables, we identified a three-state niche distribution character for the entire clade, coinciding with three major forest biomes viz. phytogeographical provinces in Madagascar: A, Northwest 'Sambirano'; B, 'Eastern Lowlands'; and C, 'Central Highlands'. A time-calibrated phylogeny and Bayesian models of niche evolution were then used to detect general trends in the direction of niche change over the clade's history (≤5.3 Ma). We found highest transitions rates between lowlands (A and B) and (mostly from B) into the highland (C), with extremely low rates out of the latter. Lowland-to-highland transitions occurred frequently during the Quaternary, suggesting that climate-induced vegetational shifts promoted niche transitions and ecological speciation at this time. CONCLUSIONS: Our results reveal that niche transitions occurred frequently and asymmetrically within this Madagascan orchid clade, and in particular over Quaternary time scales. Intrinsic features germane to Bulbophyllum (e.g., high dispersal ability, drought tolerance, multiple photosynthetic pathways) as well as extrinsic factors (ecological, historical) likely interacted to generate the niche transition patterns observed. In sum, our results support the emerging idea of dramatic environmental and climatic fluctuations in Madagascar during the recent geological past, which overturns the long-held paradigm of long-term stability in tropical forest settings. The generality of the patterns and timings reported here awaits the availability of additional comparative studies in other Madagascan endemics.

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo.

OBJECTIVE: Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types. Whereas the important role of intracellular CyPA for platelet function has been reported, the effect of extracellular CyPA on platelet function has not been investigated yet. APPROACH AND RESULTS: Inhibition of extracellular CyPA through a novel specific inhibitor MM284 reduced thrombus after ferric chloride-induced injury in vivo. In vitro extracellular CyPA enhanced thrombus formation even in CyPA(-/-) platelets. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time- and dose-dependent manner. Inhibition of the platelet surface receptor extracellular matrix metalloproteinase inducer (cluster of differentiation 147) by an anticluster of differentiation 147 monoclonal antibody significantly reduced CyPA-dependent platelet degranulation. Pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by an anticluster of differentiation 147 monoclonal antibody (intravital microscopy). The role of extracellular CyPA in adhesion could be confirmed by infusing CyPA(-/-) platelets in CyPA(+/+) mice and by infusing CyPA(+/+) platelets in CyPA(-/-) mice. Stimulation of platelets with CyPA induced phosphorylation of Akt, which could in turn be inhibited in the presence of phosphoinositid-3-kinase inhibitors. Akt-1(-/-) platelets revealed a markedly decreased degranulation on CyPA stimulation. Finally, ADP-induced platelet aggregation was attenuated by MM284, as well as by inhibiting paracrine-secreted CyPA without directly affecting Ca(2+)-signaling. CONCLUSIONS: Extracellular CyPA activates platelets via cluster of differentiation 147-mediated phosphoinositid-3-kinase/Akt-signaling, leading to enhanced adhesion and thrombus formation independently of intracellular CyPA. Targeting extracellular CyPA via a specific inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.

Thioxylated cyclosporin A for studying protein-drug interactions.

Single atom substitution of cyclosporin A (CsA) through thioxylation has been used to study the structure-activity relationship of the immunosuppressive complex, involving the CsA receptor protein cyclophilin 18 (Cyp18) and the immunological target protein phosphatase calcineurin (CaN), illustrating the contributions of peptide backbone in protein-drug interaction. Moreover, the subtle difference between thioxylation positions in CsA has led to a remarkable change in the quenching effect on Cyp18 intrinsic fluorescence. Using the thioxylated compound Cs7 as an isosteric derivative of CsA in competition assay, the experiment has led to the determination of koff value in solution. Whereas the conformational heterogeneity of CsA has been found to be associated with its two-phase binding kinetics to Cyp18, the dissociation rate of CsA from complex is independent from the initial ligand structure.

Multiple independent origins of auto-pollination in tropical orchids (Bulbophyllum) in light of the hypothesis of selfing as an evolutionary dead end.

BACKGROUND: The transition from outcrossing to selfing has long been portrayed as an 'evolutionary dead end' because, first, reversals are unlikely and, second, selfing lineages suffer from higher rates of extinction owing to a reduced potential for adaptation and the accumulation of deleterious mutations. We tested these two predictions in a clade of Madagascan Bulbophyllum orchids (30 spp.), including eight species where auto-pollinating morphs (i.e., selfers, without a 'rostellum') co-exist with their pollinator-dependent conspecifics (i.e., outcrossers, possessing a rostellum). Specifically, we addressed this issue on the basis of a time-calibrated phylogeny by means of ancestral character reconstructions and within the state-dependent evolution framework of BiSSE (Binary State Speciation and Extinction), which allowed jointly estimating rates of transition, speciation, and extinction between outcrossing and selfing. RESULTS: The eight species capable of selfing occurred in scattered positions across the phylogeny, with two likely originating in the Pliocene (ca. 4.4-3.1 Ma), one in the Early Pleistocene (ca. 2.4 Ma), and five since the mid-Pleistocene (ca. ≤ 1.3 Ma). We infer that this scattered phylogenetic distribution of selfing is best described by models including up to eight independent outcrossing-to-selfing transitions and very low rates of speciation (and either moderate or zero rates of extinction) associated with selfing. CONCLUSIONS: The frequent and irreversible outcrossing-to-selfing transitions in Madagascan Bulbophyllum are clearly congruent with the first prediction of the dead end hypothesis. The inability of our study to conclusively reject or support the likewise predicted higher extinction rate in selfing lineages might be explained by a combination of methodological limitations (low statistical power of our BiSSE approach to reliably estimate extinction in small-sized trees) and evolutionary processes (insufficient time elapsed for selfers to go extinct). We suggest that, in these tropical orchids, a simple genetic basis of selfing (via loss of the 'rostellum') is needed to explain the strikingly recurrent transitions to selfing, perhaps reflecting rapid response to parallel and novel selective environments over Late Quaternary (≤ 1.3 Ma) time scales.

Targeting Extracellular Cyclophilins Ameliorates Disease Progression in Experimental Biliary Atresia.

Biliary atresia (BA) is a devastating liver disease of unknown etiology affecting children generally within the first 3 months of life. The disease is manifested by inflammation and subsequent obstruction of the extrahepatic bile ducts, fibrosis and liver failure. The mechanisms responsible for disease pathogenesis are not fully understood, but a number of factors controlled by the SMAD signaling pathway have been implicated. In this study, we investigated the role of a known proinflammatory factor, extracellular cyclophilin A (CypA), in the pathogenesis of biliary atresia using the rhesus rotavirus (RRV) murine model. We used a unique cyclosporine A derivative, MM284, which does not enter cells and therefore inactivates exclusively extracellular cyclophilins, as a potential treatment. We demonstrated that levels of CypA in plasma of RRV-infected mice were increased significantly, and that treatment of mice with MM284 prior to or one day after disease initiation by RRV infection significantly improved the status of mice with experimental BA: weight gain was restored, bilirubinuria was abrogated, liver infiltration by inflammatory cells was reduced and activation of the SMAD pathway and SMAD-controlled fibrosis mediators and tissue inhibitor of metalloproteinases (TIMP)-4 and matrix metalloproteinase (MMP)-7 was alleviated. Furthermore, treatment of human hepatic stellate cells with recombinant cyclophilin recapitulated SMAD2/3 activation, which was also suppressed by MM284 treatment. Our data provide the first evidence that extracellular cyclophilins activate the SMAD pathway and promote inflammation in experimental BA, and suggest that MM284 may be a promising therapeutic agent for treating BA and possibly other intrahepatic chronic disorders.