Optimization of TRPV6 Calcium Channel Inhibitors Using a 3D Ligand-Based Virtual Screening Method

Herein, we report the discovery of the first potent and selective inhibitor of TRPV6, a calcium channel overexpressed in breast and prostate cancer, and its use to test the effect of blocking TRPV6-mediated Ca2+-influx on cell growth. The inhibitor was discovered through a computational method, xLOS, a 3D-shape and pharmacophore similarity algorithm, a type of ligand-based virtual screening (LBVS) method described briefly here. Starting with a single weakly active seed molecule, two successive rounds of LBVS followed by optimization by chemical synthesis led to a selective molecule with 0.3 μM inhibition of TRPV6. The ability of xLOS to identify different scaffolds early in LBVS was essential to success. The xLOS method may be generally useful to develop tool compounds for poorly characterized targets.

The Chemical Space Project

One of the simplest questions that can be asked about molecular diversity is how many organic molecules are possible in total? To answer this question, my research group has computationally enumerated all possible organic molecules up to a certain size to gain an unbiased insight into the entire chemical space. Our latest database, GDB-17, contains 166.4 billion molecules of up to 17 atoms of C, N, O, S, and halogens, by far the largest small molecule database reported to date. Molecules allowed by valency rules but unstable or nonsynthesizable due to strained topologies or reactive functional groups were not considered, which reduced the enumeration by at least 10 orders of magnitude and was essential to arrive at a manageable database size. Despite these restrictions, GDB-17 is highly relevant with respect to known molecules. Beyond enumeration, understanding and exploiting GDBs (generated databases) led us to develop methods for virtual screening and visualization of very large databases in the form of a “periodic system of molecules” comprising six different fingerprint spaces, with web-browsers for nearest neighbor searches, and the MQN- and SMIfp-Mapplet application for exploring color-coded principal component maps of GDB and other large databases. Proof-of-concept applications of GDB for drug discovery were realized by combining virtual screening with chemical synthesis and activity testing for neurotransmitter receptor and transporter ligands. One surprising lesson from using GDB for drug analog searches is the incredible depth of chemical space, that is, the fact that millions of very close analogs of any molecule can be readily identified by nearest-neighbor searches in the MQN-space of the various GDBs. The chemical space project has opened an unprecedented door on chemical diversity. Ongoing and yet unmet challenges concern enumerating molecules beyond 17 atoms and synthesizing GDB molecules with innovative scaffolds and pharmacophores.

Strengths, weaknesses, opportunities and threats: A SWOT analysis of the ecosystem services framework

The ecosystem services concept (ES) is becoming a cornerstone of contemporary sustainability thought. Challenges with this concept and its applications are well documented, but have not yet been systematically assessed alongside strengths and external factors that influence uptake. Such an assessment could form the basis for improving ES thinking, further embedding it into environmental decisions and management. The Young Ecosystem Services Specialists (YESS) completed a Strengths–Weaknesses–Opportunities–Threats (SWOT) analysis of ES through YESS member surveys. Strengths include the approach being interdisciplinary, and a useful communication tool. Weaknesses include an incomplete scientific basis, frameworks being inconsistently applied, and accounting for nature's intrinsic value. Opportunities include alignment with existing policies and established methodologies, and increasing environmental awareness. Threats include resistance to change, and difficulty with interdisciplinary collaboration. Consideration of SWOT themes suggested five strategic areas for developing and implementing ES. The ES concept could improve decision-making related to natural resource use, and interpretation of the complexities of human-nature interactions. It is contradictory – valued as a simple means of communicating the importance of conservation, whilst also considered an oversimplification characterised by ambiguous language. Nonetheless, given sufficient funding and political will, the ES framework could facilitate interdisciplinary research, ensuring decision-making that supports sustainable development.

Very Late Scaffold Thrombosis: Intracoronary Imaging and Histopathological and Spectroscopic Findings.

BACKGROUND Bioresorbable scaffolds provide transient lumen support followed by complete resorption. OBJECTIVES This study examined whether very late scaffold thrombosis (VLScT) occurs when resorption is presumed to be nearly complete. METHODS Patients with VLScT at 3 tertiary care centers underwent thrombus aspiration followed by optical coherence tomography (OCT). Thrombus aspirates were analyzed by histopathological and spectroscopic examination. RESULTS Between March 2014 and February 2015, 4 patients presented with VLScT at 44 (case 1), 19 (cases 2 and 4), and 21 (case 3) months, respectively, after implantation of an Absorb Bioresorbable Vascular Scaffold 1.1 (Abbott Laboratories, Abbott Park, Illinois). At the time of VLScT, all patients were taking low-dose aspirin, and 2 patients were also taking prasugrel. OCT showed malapposed scaffold struts surrounded by thrombus in 7.1%, 9.0%, and 8.9% of struts in cases 1, 2, and 4, respectively. Scaffold discontinuity with struts in the lumen center was the cause of malapposition in cases 2 and 4. Uncovered scaffold struts with superimposed thrombus were the predominant findings in case 3. OCT percent area stenosis at the time of VLScT was high in case 1 (74.8%) and case 2 (70.9%) without evidence of excessive neointimal hyperplasia. Spectroscopic thrombus aspirate analysis showed persistence of intracoronary polymer fragments in case 1. CONCLUSIONS VLScT may occur at advanced stages of scaffold resorption. Potential mechanisms specific for VLScT include scaffold discontinuity and restenosis during the resorption process, which appear delayed in humans; these findings suggest an extended period of vulnerability for thrombotic events.

Everolimus-eluting bioresorbable stent vs. durable polymer everolimus-eluting metallic stent in patients with ST-segment elevation myocardial infarction: results of the randomized ABSORB ST-segment elevation myocardial infarction-TROFI II trial.

AIMS Patients with ST-segment elevation myocardial infarction (STEMI) feature thrombus-rich lesions with large necrotic core, which are usually associated with delayed arterial healing and impaired stent-related outcomes. The use of bioresorbable vascular scaffolds (Absorb) has the potential to overcome these limitations owing to restoration of native vessel lumen and physiology at long term. The purpose of this randomized trial was to compare the arterial healing response at short term, as a surrogate for safety and efficacy, between the Absorb and the metallic everolimus-eluting stent (EES) in patients with STEMI. METHODS AND RESULTS ABSORB-STEMI TROFI II was a multicentre, single-blind, non-inferiority, randomized controlled trial. Patients with STEMI who underwent primary percutaneous coronary intervention were randomly allocated 1:1 to treatment with the Absorb or EES. The primary endpoint was the 6-month optical frequency domain imaging healing score (HS) based on the presence of uncovered and/or malapposed stent struts and intraluminal filling defects. Main secondary endpoint included the device-oriented composite endpoint (DOCE) according to the Academic Research Consortium definition. Between 06 January 2014 and 21 September 2014, 191 patients (Absorb [n = 95] or EES [n = 96]; mean age 58.6 years old; 17.8% females) were enrolled at eight centres. At 6 months, HS was lower in the Absorb arm when compared with EES arm [1.74 (2.39) vs. 2.80 (4.44); difference (90% CI) -1.06 (-1.96, -0.16); Pnon-inferiority <0.001]. Device-oriented composite endpoint was also comparably low between groups (1.1% Absorb vs. 0% EES). One case of definite subacute stent thrombosis occurred in the Absorb arm (1.1% vs. 0% EES; P = ns). CONCLUSION Stenting of culprit lesions with Absorb in the setting of STEMI resulted in a nearly complete arterial healing which was comparable with that of metallic EES at 6 months. These findings provide the basis for further exploration in clinically oriented outcome trials.

Arterial healing following primary PCI using the Absorb everolimus-eluting bioresorbable vascular scaffold (Absorb BVS) versus the durable polymer everolimus-eluting metallic stent (XIENCE) in patients with acute ST-elevation myocardial infarction: rationale and design of the randomised TROFI II study

AIMS The Absorb bioresorbable vascular scaffold (Absorb BVS) provides similar clinical outcomes compared with a durable polymer-based everolimus-eluting metallic stent (EES) in stable coronary artery disease patients. ST-elevation myocardial infarction (STEMI) lesions have been associated with delayed arterial healing and impaired stent-related outcomes. The purpose of the present study is to compare directly the arterial healing response, angiographic efficacy and clinical outcomes between the Absorb BVS and metallic EES. METHODS AND RESULTS A total of 191 patients with acute STEMI were randomly allocated to treatment with the Absorb BVS or a metallic EES 1:1. The primary endpoint is the neointimal healing (NIH) score, which is calculated based on a score taking into consideration the presence of uncovered and malapposed stent struts, intraluminal filling defects and excessive neointimal proliferation, as detected by optical frequency domain imaging (OFDI) six months after the index procedure. The study will provide 90% power to show non-inferiority of the Absorb BVS compared with the EES. CONCLUSIONS This will be the first randomised study investigating the arterial healing response following implantation of the Absorb BVS compared with the EES. The healing response assessed by a novel NIH score in conjunction with results on angiographic efficacy parameters and device-oriented events will elucidate disease-specific applications of bioresorbable scaffolds.

NET formation can occur independently of RIPK3 and MLKL signaling

The importance of neutrophil extracellular traps (NETs) in innate immunity is well established but the molecular mechanisms responsible for their formation are still a matter of scientific dispute. Here, we aim to characterize a possible role of the receptor-interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) signaling pathway, which are known to cause necroptosis, in NET formation. Using genetic and pharmacological approaches, we investigated whether this programmed form of necrosis is a prerequisite for NET formation. NETs have been defined as extracellular DNA scaffolds associated with the neutrophil granule protein elastase that are capable of killing bacteria. Neither Ripk3-deficient mouse neutrophils nor human neutrophils in which MLKL had been pharmacologically inactivated, exhibited abnormalities in NET formation upon physiological activation or exposure to low concentrations of PMA. These data indicate that NET formation occurs independently of both RIPK3 and MLKL signaling.

Antibody-Drug Conjugates for Tumor Targeting-Novel Conjugation Chemistries and the Promise of non-IgG Binding Proteins

Antibody-drug conjugates (ADCs) have emerged as a promising class of anticancer agents, combining the specificity of antibodies for tumor targeting and the destructive potential of highly potent drugs as payload. An essential component of these immunoconjugates is a bifunctional linker capable of reacting with the antibody and the payload to assemble a functional entity. Linker design is fundamental, as it must provide high stability in the circulation to prevent premature drug release, but be capable of releasing the active drug inside the target cell upon receptor-mediated endocytosis. Although ADCs have demonstrated an increased therapeutic window, compared to conventional chemotherapy in recent clinical trials, therapeutic success rates are still far from optimal. To explore other regimes of half-life variation and drug conjugation stoichiometries, it is necessary to investigate additional binding proteins which offer access to a wide range of formats, all with molecularly defined drug conjugation. Here, we delineate recent progress with site-specific and biorthogonal conjugation chemistries, and discuss alternative, biophysically more stable protein scaffolds like Designed Ankyrin Repeat Proteins (DARPins), which may provide such additional engineering opportunities for drug conjugates with improved pharmacological performance.

A combined biomaterial and cellular approach for annulus fibrosus rupture repair

Recurrent intervertebral disc (IVD) herniation and degenerative disc disease have been identified as the most important factors contributing to persistent pain and disability after surgical discectomy. An annulus fibrosus (AF) closure device that provides immediate closure of the AF rupture, restores disc height, reduces further disc degeneration and enhances self-repair capacities is an unmet clinical need. In this study, a poly(trimethylene carbonate) (PTMC) scaffold seeded with human bone marrow derived mesenchymal stromal cells (MSCs) and covered with a poly(ester-urethane) (PU) membrane was assessed for AF rupture repair in a bovine organ culture annulotomy model under dynamic load for 14 days. PTMC scaffolds combined with the sutured PU membrane restored disc height of annulotomized discs and prevented herniation of nucleus pulposus (NP) tissue. Implanted MSCs showed an up-regulated gene expression of type V collagen, a potential AF marker, indicating in situ differentiation capability. Furthermore, MSCs delivered within PTMC scaffolds induced an up-regulation of anabolic gene expression and down-regulation of catabolic gene expression in adjacent native disc tissue. In conclusion, the combined biomaterial and cellular approach has the potential to hinder herniation of NP tissue, stabilize disc height, and positively modulate cell phenotype of native disc tissue.

中国と湾岸を結ぶ南アジア -- パキスタン・アフガニスタンの動向と関連させて

Pakistan is geographically situated between China and the Gulf. In order to balance its strategic position against the major security threat of India, Pakistan formed a special and stable strategic alliance with China against common threats since the period of the cold war even though the two countries have neither a political ideology nor political system in common. On the other hand Pakistan established another special relation with Saudi Arabia on the basis of Islamic identity. With its expanding economic capacity, China proposed a project by the name of "new silk road economic corridor" with the intention of expanding and multiplying trade routes with the Middle East and Europe. Within this framework Pakistan is expected to expand the role of an alternative land route that connects the Gulf and China for use if unfavorable emergencies occur in the Malacca route. However, the continuous political uncertainty in Afghanistan after the pullout of US-NATO fighting forces at the end of 2014 and sporadic outbreaks of terrorist acts by Pakistan Taliban in Pakistan have increased China's anxiety regarding Uyghur issues at home. Avoiding military options for the moment, China is trying to find ways to play an active role in the security issues of Afghanistan with help from Pakistan if available. On the other hand, it is noteworthy that the Pakistani government formed in the general election of 2008 completed its full term and transferred authority to the newly elected government in 2013, something never observed before in Pakistan's history. Coincidently, in Afghanistan the presidential election was carried out peacefully in 2014 in spite of the Taliban threat. Although it is too early to make any definite conclusion, constitutional processes, in spite of their defects, reflected to some extent wishes for normal life of the people of Pakistan and Afghanistan who were disgusted with weak governance and the prevalence of terrorism.

Minor ampullate silks from Nephila and Argiope spiders: tensile properties and microstructural characterization

The mechanical behavior and microstructure of minor ampullate gland silk (miS) of two orb-web spinning species, Argiope trifasciata and Nephila inaurata, were extensively characterized, enabling detailed comparison with other silks. The similarities and differences exhibited by miS when compared with the intensively studied major ampullate gland silk (MAS) and silkworm (Bombyx mori) silk offer a genuine opportunity for testing some of the hypotheses proposed to correlate microstructure and tensile properties in silk. In this work, we show that miSs of different species show similar properties, even when fibers spun by spiders that diverged over 100 million years are compared. The tensile properties of miS are comparable to those of MAS when tested in air, significantly in terms of work to fracture, but differ considerably when tested in water. In particular, miS does not show a supercontraction effect and an associated ground state. In this regard, the behavior of miS in water is similar to that of B. mori silk, and it is shown that the initial elastic modulus of both fibers can be explained using a common model. Intriguingly, the microstructural parameters measured in miS are comparable to those of MAS and considerably different from those found in B. mori. This fact suggests that some critical microstructural information is still missing in our description of silks, and our results suggest that the hydrophilicity of the lateral groups or the large scale organization of the sequences might be routes worth exploring.

Stability and mechanical evaluation of bovine pericardium cross-linked with polyurethane prepolymer in aqueous medium

The present study investigates the potential use of non-catalyzed water-soluble blocked polyurethane prepolymer (PUP) as a bifunctional cross-linker for collagenous scaffolds. The effect of concentration (5, 10, 15 and 20%), time (4, 6, 12 and 24 h), medium volume (50, 100, 200 and 300%) and pH (7.4, 8.2, 9 and 10) over stability, microstructure and tensile mechanical behavior of acellular pericardial matrix was studied. The cross-linking index increased up to 81% while the denaturation temperature increased up to 12 °C after PUP crosslinking. PUP-treated scaffold resisted the collagenase degradation (0.167 ± 0.14 mmol/g of liberated amine groups vs. 598 ± 60 mmol/g for non-cross-linked matrix). The collagen fiber network was coated with PUP while viscoelastic properties were altered after cross-linking. The treatment of the pericardial scaffold with PUP allows (i) different densities of cross-linking depending of the process parameters and (ii) tensile properties similar to glutaraldehyde method.

The variability and interdependence of spider viscid line tensile properties

True stress-true strain curves of naturally spun viscid line fibers retrieved directly from the spiral of orb-webs built by Argiope trifasciata spiders were measured using a novel methodology. This new procedure combines a method for removing the aqueous coating of the fibers and a technique that allows the accurate measurement of their cross sectional area. Comparison of the tensile behaviour of different samples indicates that naturally spun viscid lines show a large variability, comparable to that of other silks, such as major ampullate gland silk and silkworm silk. Nevertheless, application of a statistical analysis allowed identifying two independent parameters that underlie the variability and characterize the observed range of true stress-true strain curves. Combination of this result with previous mechanical and microstructural data suggested the assignment of these two independent effects to the degree of alignment of the protein chains and to the local relative humidity which, in turn, depends on the composition of the viscous coating and on the external environmental conditions.

Material, espacio y color en Mies van der Rohe. Café Samt & Seide: Hacia una Propuesta Estructural

La aparición de varias fotografías del Café Samt & Seide no publicadas hasta 2008 en las que se observaba la luz natural de la exposición; los planos a escala de la nave donde se realizó la exposición textil; y los croquis inéditos de la instalación de la seda localizados durante el transcurso de la Tesis Doctoral por el doctorando en el plano de la Sala de Cristal, han sido las principales aportaciones de la búsqueda documental que ha tenido por objeto el conocimiento espacial e interpretación gráfica del proyecto expositivo realizada durante el transcurso de la Tesis Doctoral para poder profundizar en el alcance de la propuesta protoarquitectónica y, a través de ella, en la obra y pensamiento de Mies van der Rohe. Material, espacio y color son elementos propuestos como capítulos que estructuran la disertación y que son empleados por Mies van der Rohe como factores de una nueva expresión espacial en la exposiciones como ensayos identificados con su idea de arquitectura. En el Café Samt & Seide, Mies van der Rohe y Lilly Reich proponen una sensibilidad en el empleo del material y color trasladada al espacio mediante una atmósfera rítmica y en movimiento de superficies abstraídas e iluminadas. La estructura visual de opuestos nace del doble material textil de la exposición desplegándose en un orden espacial y constructivo de elementos independientes iniciado en los apartamentos de la Colonia Weissenhof y Sala de Cristal de Stuttgart 1926-1927, y que será trasladado desde los espacios experimentales de 1927 a las series de mármoles y cristales del Pabellón Alemán 1928-1929. La síntesis intelectual (arte-ciencia-filosofía) guía el proceso de formalización y la técnica en una intención que será trasladada a su arquitectura en el empleo de la tecnología. La creatividad y repercusión de las vanguardias en la instalación muestra la voluntad integradora de la exposición y arquitectura como arte total. Los aspectos interpretativos de las artes plásticas, visuales y musicales se integran como criterios y sensibilidades aplicadas a las estructuras yuxtapuestas material, espacial y de color. Modernidad y tradición tectónica convergen en este espacio construido cuyas superficies cubren un armazón metálico oculto. La construcción mínima textil será incorporada como idea de forma en la futura construcción del pilar metálico, la pared flotante de sus viviendas y pabellones abiertos a la naturaleza, y la gran fachada tecnológica del espacio público de sus agrupaciones en la ciudad. El espacio total del Café Samt & Seide dentro de la gran nave de Berlín anticipa las exposiciones artísticas e industriales realizadas junto a Lilly Reich en el interior de las espaciosas salas de estructura metálica como instalaciones conceptuales de gravedad y luz antecedentes de los pabellones de grandes dimensiones y espacio universal. Arquitectura, construcción y lugar son integrados en la instalación por el principio estructural como propuesta de una idea de arquitectura que es desarrollada en las exposiciones. Oscilador y bastidor son construcciones escaladas dentro de la gran nave iluminada, expresando el conjunto un orden de partes que reflejan un todo integrado en la naturaleza y lo universal. Las múltiples relaciones espaciales, constructivas y conceptuales de la exposición con su arquitectura desvelan al Café Samt & Seide como un exponente y antecedente de su obra e idea a la que definió como una propuesta estructural. La estructura, entendida como un conjunto de valores culturales y medios técnicos, es el concepto con el que Mies van der Rohe identifica la síntesis intelectualtecnológica de su tiempo, constituyendo la finalidad de su expresión material, espacial y de color. ABSTRACT The unpublished pictures of the Café Samt & Seide appeared in 2008 in which the natural light in the exhibition can be seen; the scaled plan of the nave where the textile exhibition was held; and the unpublished sketches of the silk exhibition located by the doctoral candidate while preparing his dissertation have been the main input of the data search aiming at a better understanding of the space and a graphic interpretation of the exhibition project made by the doctoral candidate during the dissertation as a basic requirement to deepen in the scope of this protoarchitectural proposal and, though it, into Mies van der Rohe’s work and thinking. Material, space and colour are the elements put forward as the chapters structuring this dissertation and were used by Mies van der Rohe as factors of a new spatial expression in expositions as tests identified with his idea of architecture. In Café Samt & Seide, Mies van der Rohe and Lilly Reich present material and colour sensitivity transferred to space though a rhythmic and moving atmosphere made up by abstracted and lit surfaces. Such a visual grammar and oppositions unfolds into a space and constructive order of independent elements originated with the apartments of the Weissenhof Colony and Glass Room in Stuttgart 1926-1927, and which were transferred these test spaces 1927 to the series of marble and glass in the German Pavilion 1928-1929. The intellectual and formal will (art-science-philosophy) guides the technical expression, which is transferred to architecture through technology. The creativity and impact of avant-gardes in the installation reflects exhibition and architecture’s will to integrate as a Total Art. The interpretative aspects of plastic, visual and musical arts are part of a sensitivity applied to the overlapped structures of material, space and colour. Tectonic modernity and tradition converge in this space built with surfaces covering a hidden metal framework. The minimal textile construction is integrated as a shape idea in his subsequent construction of the metal pillar, floating wall in his housing and pavilions open to nature, and the great technological façade of the public space of his urban clusters. The total space of the exhibition inside the great nave in Berlin anticipates the artistic and industrial exhibitions done together with Lilly Reich inside the spacious Halls built with exposed metal structures as conceptual installation of gravity and light prior to the big-dimensioned pavilions and universal space. Architecture, construction and place are integrated by the structural principle. Oscillator and framework are scaled constructions integrated inside the great, lit nave, and the whole conveys order of its parts reflecting a whole integrated in nature and the universal. The many conceptual, space and constructive link of the textile exhibition to his European and American architecture add a greater knowledge of the time when Mies van der Rohe stated he gained a “new awareness” 1926, revealing Café Samt & Seide as a valuable example and the experimental precursor of his work and idea defined by himself as a “structural proposal”. The structure, understood as a set of cultural and material values expressed by architecture, is the notion used by Mies van der Rohe to identify the intellectual-technological synthesis as the idea of a time and which is the goal of its material, space and colour expression.

Azotobacter vinelandii nitrogenase: “Kinetics of nif gene expression and insights into the roles of FdxN and NifQ in FeMo-co biosynthesis”

The Molybdenum-nitrogenase is responsible for most biological nitrogen fixation activity (BNF) in the biosphere. Due to its great agronomical importance, it has been the subject of profound genetic and biochemical studies. The Mo nitrogenase carries at its active site a unique iron-molybdenum cofactor (FeMoco) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase through a complex and highly regulated pathway involving proteins acting as molecular scaffolds, metallocluster carriers or enzymes that provide substrates in appropriate chemical forms. Specific expression regulatory factors tightly control the accumulation levels of all these other components. Insertion of FeMo-co into a P-cluster containing apo-NifDK polypeptide results in nitrogenase reconstitution. Investigation of FeMo-co biosynthesis has uncovered new radical chemistry reactions and new roles for Fe-S clusters in biology.