Broadly Applicable Research Articles

Messy eaters: Swabbing prey DNA from the exterior of inconspicuous predators when foraging cannot be observed.

Complex coevolutionary relationships among competitors, predators, and prey have shaped taxa diversity, life history strategies, and even the avian migratory patterns we see today. Consequently, accurate documentation of prey selection is often critical for understanding these ecological and evolutionary processes. Conventional diet study methods lack the ability to document the diet of inconspicuous or difficult‐to‐study predators, such as those with large home ranges and those that move vast distances over short amounts of time, leaving gaps in our knowledge of trophic interactions in many systems. Migratory raptors represent one such group of predators where detailed diet studies have been logistically challenging. To address knowledge gaps in the foraging ecology of migrant raptors and provide a broadly applicable tool for the study of enigmatic predators, we developed a minimally invasive method to collect dietary information by swabbing beaks and talons of raptors to collect trace prey DNA. Using previously published COI primers, we were able to isolate and reference gene sequences in an open‐access barcode database to identify prey to species. This method creates a novel avenue to use trace molecular evidence to study prey selection of migrating raptors and will ultimately lead to a better understanding of raptor migration ecology. In addition, this technique has broad applicability and can be used with any wildlife species where even trace amounts of prey debris remain on the exterior of the predator after feeding.

A split luciferase-based probe for quantitative proximal determination of G\u03b1q signalling in live cells

The earlier an activation of a G protein-dependent signalling cascade at a G protein-coupled receptor (GPCR) is probed, the less amplificatory effects contribute to the measured signal. This is especially useful in case of a precise quantification of agonist efficacies, and is of paramount importance, when determining agonist bias in relation to the β-arrestin pathway. As most canonical assays with medium to high throughput rely on the quantification of second messengers, and assays affording more proximal readouts are often limited in throughput, we developed a technique with a proximal readout and sufficiently high throughput that can be used in live cells. Split luciferase complementation (SLC) was applied to assess the interaction of Gαq with its effector phospholipase C-β3. The resulting probe yielded an excellent Z’ value of 0.7 and offers a broad and easy applicability to various Gαq-coupling GPCRs (hH1R, hM1,3,5R, hNTS1R), expressed in HEK293T cells, allowing the functional characterisation of agonists and antagonists. Furthermore, the developed sensor enabled imaging of live cells by luminescence microscopy, as demonstrated for the hM3R. The versatile SLC-based probe is broadly applicable e.g. to the screening and the pharmacological characterisation of GPCR ligands as well as to molecular imaging.

Developing a Broadly Applicable Measure of Risk Perception.

Decades of research identify risk perception as a largely intuitive and affective construct, in contrast to the more deliberative assessments of probability and consequences that form the foundation of risk assessment. However, a review of the literature reveals that many of the risk perception measures employed in survey research with human subjects are either generic in nature, not capturing any particular affective, probabilistic, or consequential dimension of risk; or focused solely on judgments of probability. The goal of this research was to assess a multidimensional measure of risk perception across multiple hazards to identify a measure that will be broadly useful for assessing perceived risk moving forward. Our results support the idea of risk perception being multidimensional, but largely a function of individual affective reactions to the hazard. We also find that our measure of risk perception holds across multiple types of hazards, ranging from those that are behavioral in nature (e.g., health and safety behaviors), to those that are technological (e.g., pollution), or natural (e.g., extreme weather). We suggest that a general, unidimensional measure of risk may accurately capture one's perception of the severity of the consequences, and the discrete emotions that are felt in response to those potential consequences. However, such a measure is not likely to capture the perceived probability of experiencing the outcomes, nor will it be as useful at understanding one's motivation to take mitigation action.

Development of a Broadly Applicable Assay for Measurement of Glycan-Directed Enzymatic Activity

Glycosylation is a key posttranslational modification that tags protein to membranes, organelles, secretory pathways, and degradation. Aberrant protein glycosylation is present both in acquired diseases, such as cancer and neurodegeneration, and in congenital disorders of glycosylation (CDGs). Consequently, the ability to interrogate the activity of enzymes that can modify protein glycan moieties is key for drug discovery projects aimed at finding modulators of these enzymes. To date, low-throughput technologies such as SDS-PAGE and mass spectrometry have been used, which are not suitable for compound screening in drug discovery. In the present work, a broadly applicable time-resolved fluorescence resonance energy transfer (TR-FRET) assay was developed that can determine the activity of endoglycosidase enzymes in high-throughput formats. The assay was validated using PNGaseF and EndoH as tool deglycosylases. Even though the current setup is based on the recognition of glycans that bind concanavalin A (ConA), the assay concept can be adapted to glycans that bind other lectins.

A phase resonance approach for modal testing of structures with nonlinear dissipation

The concept of nonlinear modes is useful for the dynamical characterization of nonlinear mechanical systems. While efficient and broadly applicable methods are now available for the computation of nonlinear modes, nonlinear modal testing is still in its infancy. The purpose of this work is to overcome its present limitation to conservative nonlinearities. Our approach relies on the recently extended periodic motion concept, according to which nonlinear modes of damped systems are defined as family of periodic motions induced by an appropriate artificial excitation that compensates the natural dissipation. The particularly simple experimental implementation with only a single-point, single-frequency, phase resonant forcing is analyzed in detail. The method permits the experimental extraction of natural frequencies, modal damping ratios and deflection shapes (including harmonics), for each mode of interest, as function of the vibration level. The accuracy, robustness and current limitations of the method are first demonstrated numerically. The method is then verified experimentally for a friction-damped system. Moreover, a self-contained measure for estimating the quality of the extracted modal properties is investigated. The primary advantages over alternative vibration testing methods are noise robustness, broad applicability and short measurement duration. The central limitation of the identified modal quantities is that they only characterize the system in the regime near isolated resonances.

D -Alanyl- d -Alanine Ligase as a Broad-Host-Range Counterselection Marker in Vancomycin-Resistant Lactic Acid Bacteria

ABSTRACTThe peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate and binds with high affinity to peptidoglycan ending in d-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genus Lactobacillus. Finally, our system represents the first “plug and play” counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCE The genus Lactobacillus contains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selected Lactobacillus species. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)—an essential enzyme involved in peptidoglycan synthesis—increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance in Lactobacillus; 81% of Lactobacillus species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Broad application and optimization of a single wash-step for integrated endotoxin depletion during protein purification

Endotoxins contaminate proteins that are produced in E. coli. High levels of endotoxins can influence cellular assays and cause severe adverse effects when administered to humans. Thus, endotoxin removal is important in protein purification for academic research and in GMP manufacturing of biopharmaceuticals. Several methods exist to remove endotoxin, but often require additional downstream-processing steps, decrease protein yield and are costly. These disadvantages can be avoided by using an integrated endotoxin depletion (iED) wash-step that utilizes Triton X-114 (TX114). In this paper, we show that the iED wash-step is broadly applicable in most commonly used chromatographies: it reduces endotoxin by a factor of 103 to 106 during NiNTA-, MBP-, SAC-, GST-, Protein A and CEX-chromatography but not during AEX or HIC-chromatography. We characterized the iED wash-step using Design of Experiments (DoE) and identified optimal experimental conditions for application scenarios that are relevant to academic research or industrial GMP manufacturing. A single iED wash-step with 0.75% (v/v) TX114 added to the feed and wash buffer can reduce endotoxin levels to below 2 EU/ml or deplete most endotoxin while keeping the manufacturing costs as low as possible. The comprehensive characterization enables academia and industry to widely adopt the iED wash-step for a routine, efficient and cost-effective depletion of endotoxin during protein purification at any scale.

Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization.

Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT) such as RT-qPCR and RNA-Seq. Here we describe a broadly applicable technique called ‘Ligation in situ Hybridization’ (‘LISH’), which is an alternative methodology for the analysis of FFPE RNA. LISH utilizes the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA–DNA probe pairs hybridized in situ on fixed RNA target sequences. Subsequent treatment with RNase H releases RNA-templated ligation products into solution for downstream analysis. We demonstrate several unique advantages of LISH-based assays using patient-derived FFPE tissue. These include >100-plex capability, compatibility with common histochemical stains and suitability for analysis of decade-old materials and exceedingly small microdissected tissue fragments. High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes (‘LISH-seq’), which can be amplified efficiently and with negligible bias. LISH analysis of FFPE RNA is a novel methodology with broad applications that range from multiplexed gene expression analysis to the sensitive detection of infectious organisms.

A Broadly Applicable Assay for Rapidly and Accurately Quantifying DNA Surface Coverage on Diverse Particles.

DNA-modified particles are used extensively for applications in sensing, material science, and molecular biology. The performance of such DNA-modified particles is greatly dependent on the degree of surface coverage, but existing methods for quantitation can only be employed for certain particle compositions and/or conjugation chemistries. We have developed a simple and broadly applicable exonuclease III (Exo III) digestion assay based on the cleavage of phosphodiester bonds-a universal feature of DNA-modified particles-to accurately quantify DNA probe surface coverage on diverse, commonly used particles of different compositions, conjugation chemistries, and sizes. Our assay utilizes particle-conjugated, fluorophore-labeled probes that incorporate two abasic sites; these probes are hybridized to a complementary DNA (cDNA) strand, and quantitation is achieved via cleavage and digestion of surface-bound probe DNA via Exo III's apurinic endonucleolytic and exonucleolytic activities. The presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles. Probe digestion releases a signal-generating fluorophore and liberates the intact cDNA strand to start a new cycle of hybridization and digestion, until all fluorophore tags have been released. Since the molar ratio of fluorophore to immobilized DNA is 1:1, DNA surface coverage can be determined accurately based on the complete release of fluorophores. Our method delivers accurate, rapid, and reproducible quantitation of thiolated DNA on the surface of gold nanoparticles, and also performs equally well with other conjugation chemistries, substrates, and particle sizes, and thus offers a broadly useful assay for quantitation of DNA surface coverage.

Novel small molecule modulators of plant growth and development identified by high-content screening with plant pollen.

BackgroundSmall synthetic molecules provide valuable tools to agricultural biotechnology to circumvent the need for genetic engineering and provide unique benefits to modulate plant growth and development.ResultsWe developed a method to explore molecular mechanisms of plant growth by high-throughput phenotypic screening of haploid populations of pollen cells. These cells rapidly germinate to develop pollen tubes. Compounds acting as growth inhibitors or stimulators of pollen tube growth are identified in a screen lasting not longer than 8 h high-lighting the potential broad applicability of this assay to prioritize chemicals for future mechanism focused investigations in plants. We identified 65 chemical compounds that influenced pollen development. We demonstrated the usefulness of the identified compounds as promotors or inhibitors of tobacco and Arabidopsis thaliana seed growth. When 7 days old seedlings were grown in the presence of these chemicals twenty two of these compounds caused a reduction in Arabidopsis root length in the range from 4.76 to 49.20 % when compared to controls grown in the absence of the chemicals. Two of the chemicals sharing structural homology with thiazolidines stimulated root growth and increased root length by 129.23 and 119.09 %, respectively. The pollen tube growth stimulating compound (S-02) belongs to benzazepin-type chemicals and increased Arabidopsis root length by 126.24 %.ConclusionsIn this study we demonstrate the usefulness of plant pollen tube based assay for screening small chemical compound libraries for new biologically active compounds. The pollen tubes represent an ultra-rapid screening tool with which even large compound libraries can be analyzed in very short time intervals. The broadly applicable high-throughput protocol is suitable for automated phenotypic screening of germinating pollen resulting in combination with seed germination assays in identification of plant growth inhibitors and stimulators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0875-4) contains supplementary material, which is available to authorized users.

Antifouling grafting of ceramic membranes validated in a variety of challenging wastewaters

Compared to traditional separation and purification techniques, membrane filtration is particularly beneficial for the treatment of wastewater streams such as pulp and paper mill effluents (PPME), olive oil wastewater (OOWW) and oil/gas produced water (PW). However, severe membrane fouling can be a major issue.In this work, the use of ceramic membranes and the potential for the broad applicability of a recently developed antifouling grafting was evaluated to tackle this issue. To this end, the fouling behavior of native and grafted membranes was tested in the selected difficult wastewater streams, both in dead-end and in cross-flow mode. In addition, the quality of the produced permeate water was determined to assess the overall performance of the investigated membranes for reuse or recycling of the treated wastewater.The obtained results show that grafting significantly enhances the antifouling tendency of the ceramic membranes. Particularly, the membrane grafted with methyl groups using the Grignard technique (MGR), showed in all cases no or negligible fouling as compared to the native membrane. As a consequence, the process flux or filtration capacity of the MGR membrane in cross-flow is always higher and more stable than the native membrane, even though the grafting lowers the pure water flux. Hence, the inert character of the MGR membrane is repeatedly proven and shown to be broadly applicable and generic for anti-fouling, without loss in permeate quality.Moreover, in case of OOWW, the quality of the MGR permeate is even better than that of the native membrane due to its lower fouling. All results can be explained taking into account the physico-chemical properties of foulants and membranes, as shown in previous work. In conclusion, the use of MGR membranes could provide an optimum economical solution for the treatment of the selected challenging wastewaters.

Consensus thermotolerance ranking for 110 Symbiodinium phylotypes: an exemplar utilization of a novel iterative partial‐rank aggregation tool with broad application potential

Summary Corals may reduce the effects of heat‐induced bleaching through associations with thermotolerant algal symbionts (Symbiodinium). Although hundreds of Symbiodinium genetic types (phylotypes) are known, thermotolerance has been systematically evaluated for small subsamples within individual reports, making consensus a challenging task. Data on 110 phylotypes were aggregated from 35 reports, each assessing 2–24 phylotypes (median 4). One‐third of reports include ties (phylotypes with indistinguishable thermotolerance). Between reports, most phylotypes are unique (74% assessed once), most pairwise comparisons are unknown (relative thermotolerance for 15% of the possible 5995 phylotype pairs are evaluated) and many rankings are inconsistent (52% of 58 phylotype pairs assessed in more than one report are discordantly ranked). Ranking of Symbiodinium phylotypes resulted in 64 thermotolerance cohorts (47 are single phylotypes, 17 contain 2–10 phylotypes with a median of 3 phylotypes) and indicates diverse thermotolerance capabilities within clades and among closely related phylotypes. The iterative partial‐rank aggregation method (with error estimation) introduced here is broadly applicable to any quantitative consensus rank building problem where qualitative input ranks are not fully comprehensive nor resolved. Reconstructions based on simulated data (mirroring the empirical data set) indicate robust predictive capabilities for resolving rankings (1·28 approximation of the true solution) and inferring unknown pairwise comparisons (83% correctly predicted pair rankings from only 16% known a priori). Application to Symbiodinium phylotypes represents an additional tool to generate testable hypotheses on the role of symbiont thermotolerance in an ecosystem that is collapsing in the face of climate change and provides the first quantitative index of Symbiodinium thermotolerance and its associated uncertainty. A Lay Summary is available for this article.

Development of a general copper-catalyzed vinylic Finkelstein reaction—application to the synthesis of the C1–C9 fragment of laingolide B

An efficient and broadly applicable procedure for the copper-catalyzed vinylic Finkelstein reaction is reported. Using a simple, readily available and cheap catalytic system, a broad range of alkenyl iodides and bromides can be smoothly converted to their lower homologues with high yields and full retention of the double bond geometry. Key features of this vinylic Finkelstein reaction are its broad applicability, enabling the conversion of readily available alkenyl iodides to their less available brominated and chlorinated counterparts, and the mild reaction conditions compatible with a range of highly functionalized substrates. The potential of this vinylic halogen exchange reaction in total synthesis and medicinal chemistry was demonstrated by its successful use for the synthesis of the C1–C9 fragment of laingolide B and for the late-stage modification of drug-like molecules. The extension of this halogen exchange to the acetylenic and allenic Finkelstein reactions is also reported.

Using community archetypes to better understand differential community adaptation to wildfire risk

One of the immediate challenges of wildfire management concerns threats to human safety and property in residential areas adjacent to non-cultivated vegetation. One approach for relieving this problem is to increase human community 'adaptiveness' to deal with the risk and reality of fire in a variety of landscapes. The challenge in creating 'fire-adapted communities' (FACs) is the great diversity in character and make-up of populations at risk from wildfire. This paper outlines a recently developed categorization scheme for Wildland-Urban Interface (WUI) communities based on a larger conceptual approach for understanding how social diversity is likely to influence the creation of FACs. The WUI categorization scheme situates four community archetypes on a continuum that recognizes dynamic change in human community functioning. We use results from the WUI classification scheme to outline key characteristics associated with each archetype and results from recent case studies to demonstrate the diversity across WUI communities. Differences among key characteristics of local social context will likely result in the need for different adaptation strategies to wildfire. While the WUI archetypes described here may not be broadly applicable to other parts of the world, we argue that the conceptual approach and strategies for systematically documenting local influences on wildfire adaptation have potential for broad application.This article is part of the themed issue 'The interaction of fire and mankind'.

A Broadly Applicable Method for Pd-Catalyzed Carboperfluoro-alkylation of Terminal and Internal Alkynes: A Convenient Route to Tri- and Tetrasubstituted Olefins

A three-component Pd-catalyzed difunctionalization of internal and terminal alkynes with iodoperfluoroalkanes and boronic acids is reported here. Under low catalyst loading and mild reaction conditions, perfluoroalkylated tri- and tetrasubstituted olefins were obtained in very good yields and in excellent regio- and stereoselectivities.

Photoacoustics of single laser-trapped nanodroplets for the direct observation of nanofocusing in aerosol photokinetics.

Photochemistry taking place in atmospheric aerosol droplets has a significant impact on the Earth's climate. Nanofocusing of electromagnetic radiation inside aerosols plays a crucial role in their absorption behaviour, since the radiation flux inside the droplet strongly affects the activation rate of photochemically active species. However, size-dependent nanofocusing effects in the photokinetics of small aerosols have escaped direct observation due to the inability to measure absorption signatures from single droplets. Here we show that photoacoustic measurements on optically trapped single nanodroplets provide a direct, broadly applicable method to measure absorption with attolitre sensitivity. We demonstrate for a model aerosol that the photolysis is accelerated by an order of magnitude in the sub-micron to micron size range, compared with larger droplets. The versatility of our technique promises broad applicability to absorption studies of aerosol particles, such as atmospheric aerosols where quantitative photokinetic data are critical for climate predictions.

Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo.

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small biomolecular thiols that are present in all cells and extracellular fluids of healthy mammals. It is well-known that each plays a separate, critically important role in human physiology and that abnormal levels of each are predictive of a variety of different disease states. Although a number of fluorescence-based methods have been developed that can detect biomolecules that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report, we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (NBD) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R derivative and a fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage product can undergo a further, rapid, intramolecular Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy compound (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH derivative cannot undergo a Smiles rearrangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from Cys/Hcy, respectively. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy, and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detection of GSH over Cys/Hcy in both aqueous solution and living cells.

Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

Supradentate Transmucosal Hemorrhoidopexy (Original Technical Variant).

Symptomatic hemorhoidal disease is a serious issue affecting the quality of life. The multiplicity of current interventional procedures reflects the failure of covering all pathological aspects, thus justifying the quest for other operative solutions. Any new operative procedure is challenged by the need for broad applicability and less invasive attributes and should fit inside the frame of reasonable costs, fair efficacy, low morbidity and recurrence rate based on a procedural technique that is simple enough, reproducible and easy-tolearn. Aiming to achieve these goals we conceived a procedural variant of hemohrhoidopexy fit for outpatient surgery, a technique that does not require sophisticated/costly medical equipment or disposables and is less invasive. The operative indication concerned 2nd, 3rd and 4th grade hemorrhoids. The surgical indication was represented by 2nd, 3rd and 4th grade hemorrhoids. The needed anesthesia is regional (caudal spinal anesthesia), which qualifies this procedure for private medical services. Prolapse reduction is followed by transmucosal suturing using two rows of slowly absorbable half-step staggered en-X stitches placed just above the dentate line. Preliminary results are encouraging both as therapeutic success rate and legerity of postoperative consequences. This hemorrhoidopexy is a simple and broadly applicable technique, causing mild pain, low morbidity and good efficacy, preferable to other techniques used to treat 3rd grade and selected 4th grade hemorrhoids.