Cardenolides in nectar may be more than a consequence of allocation to other plant parts: a phylogenetic study of Asclepias

The primary function of secondary plant metabolites is thought to be defence against herbivores. The frequent occurrence of these same noxious compounds in floral nectar, which functions primarily to attract pollinators, has been seen as paradoxical. Although these compounds may have an adaptive purpose in nectar, they may also occur as a nonadaptive consequence of chemical defence in other plant parts. If nectar chemistry reflects physiological constraints or passive leakage from other tissues, we expect that the identity and relative concentration of nectar cardenolides to be correlated with those of other plant parts; in contrast, discordant distributions of compounds in nectar and other tissues may suggest adaptive roles in nectar. We compared the concentrations and identities of cardenolides in the nectar, leaves and flowers of 12 species from a monophyletic clade of Asclepias. To measure putative toxicity of nectar cardenolides, we then examined the effects of a standard cardenolide (digoxin) on the behaviour of bumblebees, a common generalist pollinator of Asclepias. We found that the average cardenolide concentrations in nectar, leaves and flowers of the 12 Asclepias species were positively correlated as predicted by nonadaptive hypotheses. However, significant differences in the identities and concentrations of individual cardenolides between nectar and leaves suggest that the production or allocation of cardenolides may be independently regulated at each plant part. In addition, cardenolide concentrations in leaves and nectar exhibited no phylogenetic signal. Surprisingly, bumblebees did not demonstrate an aversion to digoxin-rich nectar, which may indicate that nectar cardenolides have little effect on pollination. Although the idea that discordant patterns of secondary metabolites across tissue types may signal adaptive functions is attractive, there is evidence to suggest constraint contributes to nectar secondary chemistry. Further work testing the ecological impacts of such patterns will be critical in determining the functional significance of nectar cardenolides

Funcionalització regioselectiva d'una cetona amb metal·lacarborans polianiònics

L’ús de metal·lacarborans en la síntesi de compostos polianiònics amb un alt contingut en bor i amb bons rendiments és un tema d’alt interès i estudi al grup de Síntesi Inorgànica i Catàlisi de l’ICMAB. Els metal·lacarborans presenten diverses aplicacions innovadores com la BNCT (Boron Neutron Capture Therapy),1 tractament d’aigües residuals,2 activitats catalítiques3 i com a agents dopants de membranes polimèriques conductores.4 Amb la perspectiva d’emprar clústers de bor per materials moleculars funcionalitzats, hem iniciat una investigació que busca la síntesi de productes híbrids amb múltiples elements electroactius (metal·lacarborans) i el grup funcional carbonil, que gràcies a la seva reactivitat coneguda ens permetrà aplicar les molècules sintetitzades a futures reaccions i aplicacions. Una de les condicions inicials plantejades en aquesta investigació és l’ús de productes químics assequibles i de gran disponibilitat, per tal de poder assegurar l’obtenció de precursors per a futures aplicacions amb bons rendiments i ràpids de sintetitzar. Per assolir aquest objectiu es van iniciar les investigacions amb la cetona més simple coneguda, l’acetona (CH3-CO-CH3) (pKa = 19.3) com a producte de partida. A partir de la reacció d’aquesta amb una base forta no nucleòfila es genera CH3-COCH2 (-). Si s’aconsegueix prevenir o minimitzar la reacció d’addició aldòlica, l’anió CH3- CO-CH2 (-) pot atacar nucleofílicament al carboni contigu al catió oxoni5 de la molècula de cobaltabisdicarballur dioxanat (cosà dioxanat), [3,3’-Co(8-(OCH2CH2)2-1,2- C2B9H10)(1’,2’-C2B9H11)], generant un derivat del cosà dioxanat que conté un grup carbonil. Aquest procediment pot ser repetit per introduir diverses unitats de metal·lacarborans (cosà, fesà, etc.), tot i que aquest procés presenta una gran dispersió de subproductes, fet que fa baixar el rendiment global i li resta interès. Un procediment molt més efectiu i que dóna un control de la regioselectivitat molt major dels centres electroactius incorporats és la síntesi acetoacètica (pKa H intercarbonílic = 10.7). Múltiples molècules homo i hetero polianiòniques amb metal·lacarborans han estat sintetitzades amb èxit en aquest treball, amb un control regioselectiu molt precís mitjançant aquest mètode sintètic, i caracteritzades per mètodes electroquímics.

Desarrollo de un método para acoplar arenos a metalacarboranos

El uso de metalacarboranos en la síntesis de compuestos aril-cobaltobis(dicarballuro) ha despertado un nuevo tema de interés y de estudio en el grupo de Síntesi Inorgànica i Catàlisi del ICMAB-CSIC. Los metalacarboranos presentan diversas aplicaciones innovadoras como la BNCT (Boron Neutron Capture Therapy), el tratamiento de aguas residuales, actividades catalíticas, como agentes dopantes en membranas poliméricas conductoras, y como integrantes en sensores potenciométricos, entre otros. El principal objetivo de este trabajo de investigación ha consistido en desarrollar un método de acoplamiento B–C sobre el anión sándwich [3,3’-Co-(1,2-C2B9H11)2]- para la formación de nuevos derivados aril-cobalto-bis(dicarballuro); éstos se han sintetizado por su posible capacidad fotoactiva. Este acoplamiento transcurre mediante una reacción de sustitución electrófila aromática (SEAr) sobre el anillo aromático o desde la óptica del metalacarborano, mediante una sustitución nucleófila inducida electrofilicamente (EINS). Dicha reacción requiere el uso de un ácido de Lewis como catalizador. El hecho que se haya utilizado AlCl3 como catalizador, hace que la reacción que se desarrolla en este trabajo recuerde de alguna manera a una reacción de Friedel-Crafts, pese a que la reacción está dirigida a la formación de un enlace B–C mediado por un ácido de Lewis. El principal problema de las reacciones de Friedel-Crafts es la elevada cantidad de areno que se precisa para llevar a cabo la reacción, debido a que el disolvente empleado puede actuar como fuente de electrófilos. El procedimiento empleado en nuestro caso utiliza el mesitileno como disolvente, el cual posee un gran impedimento estérico y un alto punto de ebullición. De esta manera, se puede realizar esta reacción utilizando entre 1.5 y 10 equivalentes de areno respecto al cobalto-bis(dicarballuro) sin que el disolvente actúe como reactivo. Se han estudiado y optimizado las condiciones experimentales para que el método sintético que genera el enlace B–C sea lo más universal posible para cualquier tipo de anillo aromático. Se han probado una gran diversidad de anillos aromáticos, desde anillos aromáticos fuertemente activados a los más desactivados, así como anillos aromáticos heterocíclicos. Finalmente, las condiciones de síntesis extraídas de este estudio son muy satisfactorias para los anillos activados probados y para los anillos débilmente desactivados. En cambio, para los arenos fuertemente desactivados, los rendimientos de la reacción han sido bajos. Por otro lado, las pruebas realizadas sobre los anillos heterocíclicos no han sido exitosas y no se ha producido el acoplamiento B–C.

Biodistribution of meglumine antimoniate in healthy and Leishmania (Leishmania) infantum chagasi-infected BALB/c mice

Pentavalent antimonials such as meglumine antimoniate (MA) are the primary treatments for leishmaniasis, a complex disease caused by protozoan parasites of the genus Leishmania . Despite over 70 years of clinical use, their mechanisms of action, toxicity and pharmacokinetics have not been fully elucidated. Radiotracer studies performed on animals have the potential to play a major role in pharmaceutical development. The aims of this study were to prepare an antimony radiotracer by neutron irradiation of MA and to determine the biodistribution of MA in healthy and Leishmania (Leishmania) infantum chagasi-infected mice. MA (Glucantime(r)) was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes, 122Sb and 124Sb, with high radionuclidic purity and good specific activity. This irradiated compound presented anti-leishmanial activity similar to that of non-irradiated MA in both in vitro and in vivo evaluations. In the biodistribution studies, healthy mice showed higher uptake of antimony in the liver than infected mice and elimination occurred primarily through biliary excretion, with a small proportion of the drug excreted by the kidneys. The serum kinetic curve was bi-exponential, with two compartments: the central compartment and another compartment associated with drug excretion. Radiotracers, which can be easily produced by neutron irradiation, were demonstrated to be an interesting tool for answering several questions regarding antimonial pharmacokinetics and chemotherapy.

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In so-called unisexual teleost fishes, a broad spectrum of evolutionaty stages with varying amounts of sexual elements has evolved. These range from pure sperm-dependent parthenogenesis (gynogenesis) without or with different amounts of paternal leakage to hybridogenesis with hemiclonal diploid gametogenesis or genome elimination folowed by meiosis (meiotic hybridogenesis). All of these phenomena are hybrid origin. Many of these fish form complexes which involve the coexistence of one or more sexually reproducing species with derived all-female forms that have various ploidy levels and reproductive modes, including gynogenesis, (meiotic) hybridogenesis nnd sexual reproduction. In teleosts, parthenogenetic reproduction is strictly dependent on sperm to initate embryonic development. As opposed to true parthenogenesis, sperm-dependent parthenogenetic teleost lineages must primarily coexist with their "sperm donor", usually males from a parental sexual lineage or from a related sexual species. In some systems, gynogens were able to escape from their initial sperm donors ("host switch") and therefore, to enlarge their ranges and ecological niches. Sperm donors normally do not contribue genetically to the next generation. However, paternal leakage is observed in many systems contributing differing amounts of genetic material (from microchromosomes to entire chromosome sets) allowing interaction between genomes of different origin. Hybridogenesis is similar to gynogenesis in depending upon coexisence with sexual species but incorporates recombined genetic material by true fertilizazion. While hybridogens usually form clonal gametes, some triploids are capable of genome elimination followed by a normal diploid meiosis. Sperm-dependent parthenogenesis and hybridogenesis combine disadvantages and advantages from both sexuality and asexuality. Here, we give an overview of sperm-dependent breeding complexes in fishes, discuss the evolutionary consequences of paternal leakage, and speculate about the evolutionary significance of intergenomic (re)combination.

Malignant pleural mesothelioma: leukocyte recruitment is not required for drug delivery induced by photodynamic therapy in a human xenograft model

Objective: The pre-treatment of tumor neo-vessels by photodynamic therapy (PDT) was shown to improve the distribution of chemotherapy administered subsequently. However, the precise mechanism by which PDT modifies the tumor vasculature is unknown. We have recently shown that leukocyteendothelial cell interaction was essential for PDT induced drug delivery to normal tissue. Our purpose was to determine if PDT could enhance drug distribution in malignant mesothelioma and if a comparable role for leucocytes existed.Methods: We grew human mesothelioma xenografts (H-meso-1) in the dorsal skinfold chambers of nude mice (n = 28). The rolling, sticking and recruitment of leucocytes was assessed in tumor and normal vessels following PDT (Visudyne 0?4 mg/kg, fluence rate 200 mW/cm2, fluence 60 J/cm2) using intravital microscopy. In parallel, the distribution of a macromolecule (FITC dextran, 2000 kDa) administered after PDT was determined. We compared these variables in control (no PDT), PDT + IgG (non specific antibody) and PDT + pan-selectin antibody (monoclonal P-E-L selectin antibody).Results: PDT significantly enhanced the distribution of FITC dextran in mesothelioma xenografts compared to controls. Interestingly, PDT enhanced the leukocyte-endothelial interaction significantly (rolling and recruitment)in tumor and surrounding normal vessels compared to controls. Leukocyte recruitment was significantly down-regulated by pan-selectin antibodies in tumor tissues. However, the suppression of leucocyte recruitement did not affect the extravasation of FITC-dextran in tumor tissue.Conclusion:PDTpre-treatment of the mesothelioma vasculature can enhance the distribution of macromolecular drugs administered subsequently. However, unlike normal vessels, leukocyte-endothelial cell interaction is not required for PDT induced leakage.

Combined laparoscopic-endoscopic method using an omental plug for therapy of gastroduodenal ulcer perforation.

BACKGROUND: Laparoscopic techniques have been proposed as an alternative to open surgery for therapy of peptic ulcer perforation. They provide better postoperative comfort and absence of parietal complications, but leakage occurs in 5% of cases. We describe a new method combining laparoscopy and endoluminal endoscopy, designed to ensure complete closure of the perforation. METHODS: Six patients with anterior ulcer perforations (4 duodenal, 2 gastric) underwent a concomitant laparoscopy and endoluminal endoscopy with closure of the orifice by an omental plug attracted into the digestive tract. RESULTS: All perforations were sealed. The mean operating time was 72 minutes. The mean hospital stay was 5.5 days. There was no morbidity and no mortality. At the 30-day evaluation all ulcers but one (due to Helicobacter pylori persistence) were healed. CONCLUSIONS: This method is safe and effective. Its advantages compared with open surgery or laparoscopic patching as well as its cost-effectiveness should be studied in prospective randomized trials.

The role of potassium in inflammasome activation by bacteria.

Many Gram-negative bacteria possess a type III secretion system (TTSS( paragraph sign)) that can activate the NLRC4 inflammasome, process caspase-1 and lead to secretion of mature IL-1beta. This is dependent on the presence of intracellular flagellin. Previous reports have suggested that this activation is independent of extracellular K(+) and not accompanied by leakage of K(+) from the cell, in contrast to activation of the NLRP3 inflammasome. However, non-flagellated strains of Pseudomonas aeruginosa are able to activate NLRC4, suggesting that formation of a pore in the cell membrane by the TTSS apparatus may be sufficient for inflammasome activation. Thus, we set out to determine if extracellular K(+) influenced P. aeruginosa inflammasome activation. We found that raising extracellular K(+) prevented TTSS NLRC4 activation by the non-flagellated P. aeruginosa strain PA103DeltaUDeltaT at concentrations above 90 mm, higher than those reported to inhibit NLRP3 activation. Infection was accompanied by efflux of K(+) from a minority of cells as determined using the K(+)-sensitive fluorophore PBFI, but no formation of a leaky pore. We obtained exactly the same results following infection with Salmonella typhimurium, previously described as independent of extracellular K(+). The inhibitory effect of raised extracellular K(+) on NLRC4 activation thus reflects a requirement for a decrease in intracellular K(+) for this inflammasome component as well as that described for NLRP3.

Sodium sensing in neurons with a dendrimer-based nanoprobe.

Ion imaging is a powerful methodology to assess fundamental biological processes in live cells. The limited efficiency of some ion-sensing probes and their fast leakage from cells are important restrictions to this approach. In this study, we present a novel strategy based on the use of dendrimer nanoparticles to obtain better intracellular retention of fluorescent probes and perform prolonged fluorescence imaging of intracellular ion dynamics. A new sodium-sensitive nanoprobe was generated by encapsulating a sodium dye in a PAMAM dendrimer nanocontainer. This nanoprobe is very stable and has high sodium sensitivity and selectivity. When loaded in neurons in live brain tissue, it homogenously fills the entire cell volume, including small processes, and stays for long durations, with no detectable alterations of cell functional properties. We demonstrate the suitability of this new sodium nanosensor for monitoring physiological sodium responses such as those occurring during neuronal activity.

Establishment of a reliable laser-Induced Choroidal Neovascularization Animal Model

Purpose: Pathologic choroidal neovascularizations (CNV) are implicated in the wet form of age-related macular degeneration (ARMD). Abnormal vessel growth is also observed in disease when hypoxia and/or inflammation occur. Our goal is to establish a standard protocol of laser-induced CNV in mice that have different levels of pigmentation to identify the most reliable animal model.Methods: CNV was induced by 4 burns around the optic disk, using a green argon laser (100μm diameter spot size; 0,05 sec. duration) in C57/Bl6, DBA/1 and Balb/c to ascertain the efficacy of the method in function of retina pigmentation. Five different intensities were tested and Bruch's membrane disruption was identified by the appearance of a bubble at the site of photocoagulation. Fluorescein angiographies (FA) were undertaken 14 days post lesion and CNV area was quantified by immunohistochemistry on cryosections.Results: CNV retina area was related to spot intensity after laser injury. While 180mW and 200mW do not induce reliable CNV (respectively 27.85±0.35% and 29±1.67% of the retina surface), 260mW is required to induce 51,07±8.52% of CNV in C57/Bl6 mice. For the DBA/1 strain, less pigmented, 200mW was sufficient to induce 49.35±3.9% of CNV, indicating that lower intensity are required to induce CNV. Furthermore, an intensity of 180mW induced greater CNV (35.55±6.01%) than in C57/Bl6 mice. Nevertheless, laser did not induce reproducible 50% CNV in Balb/c albino mice for all intensities tested. Isolectin-B4 and GFAP stainings revealed neovessel formation and photoreceptor (PR) degeneration at the impact site. The presence of glia was observed throughout all the retinal layers and angiograms showed fluorescein leakage in pigmented mice.Conclusions: The establishment of a standard protocol to induce CNV and subsequent PR degeneration is of prime importance for the use of the laser-induced CNV model and will allow to evaluate the therapeutic potency of agents to prevent CNV and retinal degeneration.

A Comparative Study of Transvaginal Tape, Transobturator Tape Outside-in, and Transvaginal Tape-obturator Inside-out Surgical Procedures in the Treatment of Stress Urinary Incontinence

The aim of this work was to use the Urinary Distress Inventory (UDI-6) and Incontinence Impact Quality of Life (IIQ-7) questionnaires to compare 3 surgical techniques for stress urinary incontinence: the transvaginal tape (TVT) (105 women), the transobturator tape outside-in (TOT) (43 women), and the transvaginal tape-obturator inside-out (TVT-O) (54 women). There were no significant differences in frequent urination, urine leakage related to the feeling of urgency, urine leakage related to physical activity, or small amounts of urine leakage. TVT-operated women had a lower percentage of micturition difficulties compared with TOT women. TVT-O-operated women described slight discomfort in the genital area compared with the TVT technique, but this difference was not significant when compared with the TOT technique. When utilizing the UDI-6 and IIQ-7 scoring modifications before and after surgery, no difference among these 3 techniques is apparent.

Multi-layer amniotic membrane (MLAM) transplantation for non traumatic corneal perforations or descemetoceles

Purpose:To describe the indications, the surgical procedure and the clinical outcome of MLAM in the treatment of non traumatic corneal perforations and descemetoceles . Methods:A prospective, non comparative, interventional case series of eight consecutive patients (mean age 59 years old, 6 men and 2 women) with non traumatic corneal perforations or descemetoceles.The surgery consisted in a MLAM transplantation of a cryopreservated human amniotic membrane. The series included: three active herpetic keratitis, one rosacea, one perforation of an hydrops, one cicatricial pemphigoid, one perforation after an abcess in a corneal graft and one perforation after protonbeamtherapy. The clinical outcome included: the follow-up, the integrity of the eye, corneal epithelialization, inflammation and neovascularization, and the integration of the MLAM. Stromal thickness was followed precisely with the slit lamp. A corneal graft was performed at one patient after the MLAM, allowing microscopic investigation of the removed MLAM integrated in the cornea. Results:The mean follow-up was 8.78 months (range 3.57 to 30.17). Amniotic membrane transplantation was successful and reduced inflammation in 7 patients out of 8 ,after one procedure.One patient who presented a large herpetic keratitis epithelial defect with corneal anaesthesia had his MLAM dissolved after two weeks with an aqueous leakage. Epithelium healed within 3 weeks above 7 MLAM and remained stable at 3 months in 7 out of 8 patients. MLAM opacification gradually disappeared over a few months, however, stromal layers filling in the corneal perforations or above the descemetoceles remained stable. Conclusions:MLAM transplantation is a safe, effective and useful technique to cure non traumatic corneal perforations and descemetoceles. It can be performed in emergency despite the presence of an active inflammation or infection. By facilitating epithelialization, reducing inflammation and neovascularization, it allows corneal surface reconstruction in patients with persistent epithelial defects and corneal melting that usually ends in a perforation. For full visual rehabilitation, a delayed penetrating keratoplasty is required.

A prospective randomised double-blind controlled trial evaluating the effect of trans-sacral magnetic stimulation in women with overactive bladder.

Overactive bladder (OAB) is a prevalent condition with 16% of adults having one or more symptoms that significantly affect quality of life. Transcutaneous electrical nerve stimulation and neuromodulators have had success in treating OAB but are expensive, invasive, and sometimes cumbersome. We developed an alternative neuromodulatory technique that involves electromagnetic stimulation of the sacral nerve roots with a portable electromagnetic device to produce trans-sacral stimulation of the S3 and S4 sacral nerve roots. The aim of this study was to evaluate the impact of this device on OAB symptoms in women with a prospectively randomised double-blind controlled study. Following a power analysis, women with symptoms of OAB were prospectively recruited with ethical approval for randomisation to an active treatment (n = 33) or placebo group (n = 30) in a double-blind trial. The patient, at home, used the belt device daily for 20 min over 12 weeks. Outcome measures included a 3-day voiding diary, 1 h pad test, visual analogue score (VAS) for symptom impact (0-100%), Kings Health Questionnaire (KHQ) and Australian Quality of Life questionnaire (AQOL) at baseline, 6 and 12 weeks. Overall, no difference was found between groups for any of the research questions. Specifically, we were unable to demonstrate any difference between the active and sham device groups in frequency, nocturia, urinary leakage, or quality of life, nor was there any evidence of a placebo effect. The quality of the data was high with the number of missing observations (especially for disease specific KHQ and general AQOL) being few. This attempt to promote trans-sacral electromagnetic neuromodulation with a specially created device was ineffective on the symptoms of OAB.

Charge dependent substrate activity of C3' and N3 functionalized, organometallic technetium and rhenium-labeled thymidine derivatives toward human thymidine kinase 1.

Human cytosolic thymidine kinase (hTK1) has proven to be a suitable target for the noninvasive imaging of cancer cell proliferation using radiolabeled thymidine analogues such as [(18)F]3'-fluoro-3'-deoxythymidine ([(18)F]FLT). A thymidine analogue for single photon emission computed tomography (SPECT), which incorporates the readily available and inexpensive nuclide technetium-99m, would be of considerable practical interest. hTK1 is known to accommodate modification of the structure of the natural substrate thymidine at the positions N3 and C3' and, to a lesser extent, C5. In this work, we used the copper-catalyzed azide-alkyne cycloaddition to synthesize two series of derivatives in which thymidine is functionalized at either the C3' or N3 position with chelating systems suitable for the M(CO)(3) core (M = (99m)Tc, Re). The click chemistry approach enabled complexes with different structures and overall charges to be synthesized from a common precursor. Using this strategy, the first organometallic hTK1 substrates in which thymidine is modified at the C3' position were identified. Phosphorylation of the organometallic derivatives was measured relative to thymidine. We have shown that the influence of the overall charge of the derivatives is dependent on the position of functionalization. In the case of the C3'-functionalized derivatives, neutral and anionic substrates were most readily phosphorylated (20-28% of the value for the parent ligand thymidine), whereas for the N3-functionalized derivatives, cationic and neutral complexes were apparently better substrates for the enzyme (14-18%) than anionic derivatives (9%).

New ideas need new space

We develop a setting with weak intellectual property rights, where firms' boundaries, location and knowledge spillovers are endogenous. We have two main results. The first one is that, if communication costs increase with distance, entrepreneurs concerned about information leakage have a benefit from locating away from the industry center: distance is an obstacle to collusive trades between members andnon-members. The second result is that we identify a trade-off for the entrepreneur between owning a facility (controlling all its characteristics) and sharing a facility with a {\it non-member} (an agent not involved in production), therefore losing control over some of its characteristics. We focus on ``location" as the relevant characteristic of the facility, but location can be used as a spatial metaphor for other relevant characteristics of the facility. For theentrepreneur, sharing the facility with non-members implies that the latter, as co-owners, know the location (even if they do not have access to it). Knowledge of the location for the co-owners facilitates collusion with employees, what increases leakage. The model yields a benefit for new plants from spatial dispersion (locating at the periphery of the industry), particularly so for new plants of new firms.We relate this result with recent empirical findings on the dynamics of industry location.