MALDI-TOF-MS-Based Identification of Monoclonal Murine Anti-SARS-CoV-2 Antibodies within One Hour.

During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 °C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.

A broadly cross-reactive monoclonal antibody against hepatitis E virus capsid antigen.

To generate a hepatitis E virus (HEV) genotype 3 (HEV-3)-specific monoclonal antibody (mAb), the Escherichia coli-expressed carboxy-terminal part of its capsid protein was used to immunise BALB/c mice. The immunisation resulted in the induction of HEV-specific antibodies of high titre. The mAb G117-AA4 of IgG1 isotype was obtained showing a strong reactivity with the homologous E. coli, but also yeast-expressed capsid protein of HEV-3. The mAb strongly cross-reacted with ratHEV capsid protein derivatives produced in both expression systems and weaker with an E. coli-expressed batHEV capsid protein fragment. In addition, the mAb reacted with capsid protein derivatives of genotypes HEV-2 and HEV-4 and common vole hepatitis E virus (cvHEV), produced by the cell-free synthesis in Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cell lysates. Western blot and line blot reactivity of the mAb with capsid protein derivatives of HEV-1 to HEV-4, cvHEV, ratHEV and batHEV suggested a linear epitope. Use of truncated derivatives of ratHEV capsid protein in ELISA, Western blot, and a Pepscan analysis allowed to map the epitope within a partially surface-exposed region with the amino acid sequence LYTSV. The mAb was also shown to bind to human patient-derived HEV-3 from infected cell culture and to hare HEV-3 and camel HEV-7 capsid proteins from transfected cells by immunofluorescence assay. The novel mAb may serve as a useful tool for further investigations on the pathogenesis of HEV infections and might be used for diagnostic purposes. KEY POINTS: • The antibody showed cross-reactivity with capsid proteins of different hepeviruses. • The linear epitope of the antibody was mapped in a partially surface-exposed region. • The antibody detected native HEV-3 antigen in infected mammalian cells.

emEubostrichopsis/em emjohnpearsei/em n. gen., n. sp., the first stilbonematid nematode (Nematoda, Desmodoridae) from the US West Coast.

A new genus of the marine Stilbonematinae (Nematoda, Desmodoridae) is described from the Pacific coast of the United States of America. The worms inhabit the sulfidic sediment among the roots of the surfgrass Phyllospadix sp. in the rocky intertidal. The ectosymbiotic coat is of a new type for Stilbonematinae. It consists of rod-shaped bacteria pointed at both poles densely attached with one pole to the host cuticle. This is the first report of this symbiotic nematode subfamily from the US West Coast.

On the origin of the widespread self-compatible allotetraploid Capsella bursa-pastoris (Brassicaceae).

Polyploidy, or whole-genome duplication, is a common speciation mechanism in plants. An important barrier to polyploid establishment is a lack of compatible mates. Because self-compatibility alleviates this problem, it has long been hypothesized that there should be an association between polyploidy and self-compatibility (SC), but empirical support for this prediction is mixed. Here, we investigate whether the molecular makeup of the Brassicaceae self-incompatibility (SI) system, and specifically dominance relationships among S-haplotypes mediated by small RNAs, could facilitate loss of SI in allopolyploid crucifers. We focus on the allotetraploid species Capsella bursa-pastoris, which formed ~300 kya by hybridization and whole-genome duplication involving progenitors from the lineages of Capsella orientalis and Capsella grandiflora. We conduct targeted long-read sequencing to assemble and analyze eight full-length S-locus haplotypes, representing both homeologous subgenomes of C. bursa-pastoris. We further analyze small RNA (sRNA) sequencing data from flower buds to identify candidate dominance modifiers. We find that C. orientalis-derived S-haplotypes of C. bursa-pastoris harbor truncated versions of the male SI specificity gene SCR and express a conserved sRNA-based candidate dominance modifier with a target in the C. grandiflora-derived S-haplotype. These results suggest that pollen-level dominance may have facilitated loss of SI in C. bursa-pastoris. Finally, we demonstrate that spontaneous somatic tetraploidization after a wide cross between C. orientalis and C. grandiflora can result in production of self-compatible tetraploid offspring. We discuss the implications of this finding on the mode of formation of this widespread weed.

Vapochromism and Magnetochemical Switching of a Nickel(II) Paddlewheel Complex by Reversible NH3 Uptake and Release.

Reaction of [NiCl2 (PnH)4 ] (1) (PnH=6-tert-butyl-pyridazine-3-thione) with NiCl2 affords the binuclear paddlewheel (PW) complex [Ni2 (Pn)4 ] (2). Diamagnetic complex 2 is the first example of a PW complex capable of reversibly binding and releasing NH3 . The NH3 ligand in [Ni2 (Pn)4 (NH3 )] (2⋅NH3 ) enforces major spectroscopic and magnetic susceptibility changes, thus displaying vapochromic properties (λmax (2)=532 nm, λmax (2⋅NH3 )=518 nm) and magnetochemical switching (2: S=0; 2⋅NH3 : S=1). Upon repeated adsorption/desorption cycles of NH3 the PW core remains intact. Compound 2 can be embedded into thin polyurethane films (2P ) under retention of its sensing abilities. Therefore, 2 qualifies as reversible optical probe for ammonia. The magnetochemical switching of 2 and 2⋅NH3 was studied in detail by SQUID measurements showing that in 2⋅NH3 , solely the Ni atom coordinated the NH3 molecule is responsible for the paramagnetic behavior.

A retrospective multicenter study on the use of locking compression plates for scapulohumeral arthrodesis in small equids.

OBJECTIVE: To report on technical aspects and outcomes after scapulohumeral arthrodesis (SHA) with a locking compression plate (LCP) in small equids. STUDY DESIGN: Retrospective multicenter case series. ANIMALS: Client-owned Shetland Ponies, Miniature Shetlands, and American Miniature Horses (n = 15). METHODS: Inclusion criteria were completed SHA with an LCP to treat scapulohumeral osteoarthritis/subluxation and availability of postoperative radiographs. Contributing surgeons completed a questionnaire to collect data. All radiographs were reviewed for this study. Follow-up information was obtained via re-admission to the hospital or telephone interview of the referring veterinarian or owner. Outcome was subjectively scored as excellent, good, moderate, or poor based on lameness and function. RESULTS: Sixteen SHAs were performed in 15 equids (body weight 65-145 kg) by nine different surgeons. A narrow 4.5/5.0 mm LCP (7-16 holes) was used in 14 of the 16 SHAs. Follow-up was collected 2.5 years after surgery (median; range: 9 weeks to 10 years). Major complications related to the SHAs were noted in 4 of the 13 horses alive long term, consisting of implant failure (n = 1), surgical site infection (n = 2), and scapular fracture (n = 1). The outcome was graded as excellent in 4, good in 5, and moderate in 4 horses. Two ponies were euthanized because scapulohumeral osteoarthritis and subluxation developed in the contralateral limb. CONCLUSION: Although major complications occurred in about one-third of ponies, SHA with LCP led to long-term survival with good function in most ponies. CLINICAL SIGNIFICANCE: SHA with an LCP offers a high chance for good long-term outcome in small equids although contralateral disease may affect outcome.

Assumptions in ecosystem service assessments: Increasing transparency for conservation.

Conservation efforts are increasingly supported by ecosystem service assessments. These assessments depend on complex multi-disciplinary methods, and rely on a number of assumptions which reduce complexity. If assumptions are ambiguous or inadequate, misconceptions and misinterpretations may arise when interpreting results of assessments. An interdisciplinary understanding of assumptions in ecosystem service science is needed to provide consistent conservation recommendations. Here, we synthesise and elaborate on 12 prevalent types of assumptions in ecosystem service assessments. These comprise conceptual and ethical foundations of the ecosystem service concept, assumptions on data collection, indication, mapping, and modelling, on socio-economic valuation and value aggregation, as well as about using assessment results for decision-making. We recommend future assessments to increase transparency about assumptions, and to test and validate them and their potential consequences on assessment reliability. This will support the taking up of assessment results in conservation science, policy and practice.

[Value of dual-energy computed tomography for detection of left atrial appendage thrombus]. / Mehrwert der Dual-Energy-Computertomographie zur Detektion von Thromben des linken Vorhofohrs.

OBJECTIVE: Contrast-enhanced computed tomography (CT) is a convenient method to visualize left atrial appendage (LAA) thrombi. We determined whether diagnostic accuracy improves by including dual-energy as compared to transesophageal echocardiography (TEE). Furthermore, the influence of protocol parameters on radiation dose were quantified. METHODS: Patients were assigned to the different CT protocols. All CTs were assessed qualitatively for presence of LAA thrombi and dual-energy CT scans quantitatively for iodine concentration. TEE was assessed qualitatively for the presence of thrombi. RESULTS: Of 32 enrolled patients, 6 had a thrombus in TEE. Qualitative CT assessment yielded 83% sensitivity and 88% specificity. In the 26 patients who underwent dual-energy CT, median iodine concentration was 8.6 mg/cm3 and significantly lower in patients with than without LAA thrombi ; furthermore, it provided value for detecting LAA thrombi (AUC: 0.950 vs 0.867 for combined vs. only qualitative assessment, p = 0.04). The median radiation dose was 1.83 mSv; independently lower in scanning only LAA and with prospective gating , while arrhythmia and dual-energy did not contribute independently. CONCLUSION: CT provides good diagnostic accuracy for detecting LAA thrombi, which can further be improved if iodine density measurements by dual-energy are incorporated. With an optimized protocol, reasonably low radiation dose can be achieved.

Shared Socio-economic Pathways for European agriculture and food systems: The Eur-Agri-SSPs.

Scenarios describe plausible and internally consistent views of the future. They can be used by scientists, policymakers and entrepreneurs to explore the challenges of global environmental change given an appropriate level of spatial and sectoral detail and systematic development. We followed a nine-step protocol to extend and enrich a set of global scenarios - the Shared Socio-economic Pathways (SSPs) - providing regional and sectoral detail for European agriculture and food systems using a one-to-one nesting participatory approach. The resulting five Eur-Agri-SSPs are titled (1) Agriculture on sustainable paths, (2) Agriculture on established paths, (3) Agriculture on separated paths, (4) Agriculture on unequal paths, and (5) Agriculture on high-tech paths. They describe alternative plausible qualitative evolutions of multiple drivers of particular importance and high uncertainty for European agriculture and food systems. The added value of the protocol-based storyline development process lies in the conceptual and methodological transparency and rigor; the stakeholder driven selection of the storyline elements; and consistency checks within and between the storylines. Compared to the global SSPs, the five Eur-Agri-SSPs provide rich thematic and regional details and are thus a solid basis for integrated assessments of agriculture and food systems and their response to future socio-economic and environmental changes.

Isomers in chlorido and alkoxido-substituted oxidorhenium(v) complexes: effects on catalytic epoxidation activity.

The syntheses and characterizations of oxidorhenium(v) complexes trans-dichlorido [ReOCl2(PPh3)(L1a)] (trans-2a), cis-dichlorido [ReOCl2(PPh3)(L1b)] (cis-2b) and ethoxido-complex [ReO(OEt)(L1b)2] (4b), ligated with the dimethyloxazoline-phenol ligands HL1a and HL1b are described. The bidentate ligand HL1a (2-(4,4-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)-phenol) is unsubstituted on the phenol ring; ligand HL1b (2-(4,4-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)-4-nitrophenol) contains a nitro group in para-position to the hydroxy group. In the reaction of precursor complex [ReOCl3(PPh3)2] and HL1a the two stereoisomers cis/trans-2a, with respect to chlorido ligands, are formed. The solid state structures of both isomers cis- and trans-2a were determined by single crystal X-ray diffraction analysis. In contrast, with ligand HL1b, only the cis-isomer cis-2b was obtained. Ethoxido-complex 4b is exclusively obtained when precursor [ReOCl3(OPPh3)(SMe2)] is reacted with 2 equiv. of HL1b in ethanol in the presence of the base 2,6-dimethylpyridine (lutidine). If no lutidine is added, chlorido-complex [ReOCl(L1b)2] (3b) is obtained. Complexes [ReOCl2(PPh3)(L1a)] (cis/trans-2a), [ReOCl2(PPh3)(L1b)] (cis-2b), [ReO(OMe)(L1a)2] (4a) and [ReO(OEt)(L1b)2] (4b) were tested as homogeneous catalysts in the benchmark reaction of cyclooctene epoxidation. The influence of isomerism and effects of ligand substitutions on catalytic activity was investigated. Based on the time-conversion plots it can be concluded that cis/trans-isomerism does not influence catalytic activity, but electron-withdrawing substituents, as in cis-2b, 3b and 4b, show a beneficial effect.

Morphology of obligate ectosymbionts reveals Paralaxus gen. nov.: A new circumtropical genus of marine stilbonematine nematodes.

Stilbonematinae are a subfamily of conspicuous marine nematodes, distinguished by a coat of sulphur-oxidizing bacterial ectosymbionts on their cuticle. As most nematodes, the worm hosts have a relatively simple anatomy and few taxonomically informative characters, and this has resulted in numerous taxonomic reassignments and synonymizations. Recent studies using a combination of morphological and molecular traits have helped to improve the taxonomy of Stilbonematinae but also raised questions on the validity of several genera. Here, we describe a new circumtropically distributed genus Paralaxus (Stilbonematinae) with three species: Paralaxus cocos sp. nov., P. bermudensis sp. nov. and P. columbae sp. nov. We used single worm metagenomes to generate host 18S rRNA and cytochrome c oxidase I (COI) as well as symbiont 16S rRNA gene sequences. Intriguingly, COI alignments and primer matching analyses suggest that the COI is not suitable for PCR-based barcoding approaches in Stilbonematinae as the genera have a highly diverse base composition and no conserved primer sites. The phylogenetic analyses of all three gene sets, however, confirm the morphological assignments and support the erection of the new genus Paralaxus as well as corroborate the status of the other stilbonematine genera. Paralaxus most closely resembles the stilbonematine genus Laxus in overlapping sets of diagnostic features but can be distinguished from Laxus by the morphology of the genus-specific symbiont coat. Our re-analyses of key parameters of the symbiont coat morphology as character for all Stilbonematinae genera show that with amended descriptions, including the coat, highly reliable genus assignments can be obtained.

A protocol to develop Shared Socio-economic Pathways for European agriculture.

Moving towards a more sustainable future requires concerted actions, particularly in the context of global climate change. Integrated assessments of agricultural systems (IAAS) are considered valuable tools to provide sound information for policy and decision-making. IAAS use storylines to define socio-economic and environmental framework assumptions. While a set of qualitative global storylines, known as the Shared Socio-economic Pathways (SSPs), is available to inform integrated assessments at large scales, their spatial resolution and scope is insufficient for regional studies in agriculture. We present a protocol to operationalize the development of Shared Socio-economic Pathways for European agriculture - Eur-Agri-SSPs - to support IAAS. The proposed design of the storyline development process is based on six quality criteria: plausibility, vertical and horizontal consistency, salience, legitimacy, richness and creativity. Trade-offs between these criteria may occur. The process is science-driven and iterative to enhance plausibility and horizontal consistency. A nested approach is suggested to link storylines across scales while maintaining vertical consistency. Plausibility, legitimacy, salience, richness and creativity shall be stimulated in a participatory and interdisciplinary storyline development process. The quality criteria and process design requirements are combined in the protocol to increase conceptual and methodological transparency. The protocol specifies nine working steps. For each step, suitable methods are proposed and the intended level and format of stakeholder engagement are discussed. A key methodological challenge is to link global SSPs with regional perspectives provided by the stakeholders, while maintaining vertical consistency and stakeholder buy-in. We conclude that the protocol facilitates systematic development and evaluation of storylines, which can be transferred to other regions, sectors and scales and supports inter-comparisons of IAAS.

Genetic basis and timing of a major mating system shift in Capsella.

A crucial step in the transition from outcrossing to self-fertilization is the loss of genetic self-incompatibility (SI). In the Brassicaceae, SI involves the interaction of female and male specificity components, encoded by the genes SRK and SCR at the self-incompatibility locus (S-locus). Theory predicts that S-linked mutations, and especially dominant mutations in SCR, are likely to contribute to loss of SI. However, few studies have investigated the contribution of dominant mutations to loss of SI in wild plant species. Here, we investigate the genetic basis of loss of SI in the self-fertilizing crucifer species Capsella orientalis, by combining genetic mapping, long-read sequencing of complete S-haplotypes, gene expression analyses and controlled crosses. We show that loss of SI in C. orientalis occurred < 2.6 Mya and maps as a dominant trait to the S-locus. We identify a fixed frameshift deletion in the male specificity gene SCR and confirm loss of male SI specificity. We further identify an S-linked small RNA that is predicted to cause dominance of self-compatibility. Our results agree with predictions on the contribution of dominant S-linked mutations to loss of SI, and thus provide new insights into the molecular basis of mating system transitions.

Dioxygen Activation with Molybdenum Complexes Bearing Amide-Functionalized Iminophenolate Ligands.

Two novel iminophenolate ligands with amidopropyl side chains (HL2 and HL3) on the imine functionality have been synthesized in order to prepare dioxidomolybdenum(VI) complexes of the general structure [MoO2L2] featuring pendant internal hydrogen bond donors. For reasons of comparison, a previously published complex featuring n-butyl side chains (L1) was included in the investigation. Three complexes (1-3) obtained using these ligands (HL1-HL3) were able to activate dioxygen in an in situ approach: The intermediate molybdenum(IV) species [MoO(PMe3)L2] is first generated by treatment with an excess of PMe3. Subsequent reaction with dioxygen leads to oxido peroxido complexes of the structure [MoO(O2)L2]. For the complex employing the ligand with the n-butyl side chain, the isolation of the oxidomolybdenum(IV) phosphino complex [MoO(PMe3)(L1)2] (4) was successful, whereas the respective Mo(IV) species employing the ligands with the amidopropyl side chains were found to be not stable enough to be isolated. The three oxido peroxido complexes of the structure [MoO(O2)L2] (9-11) were systematically compared to assess the influence of internal hydrogen bonds on the geometry as well as the catalytic activity in aerobic oxidation. All complexes were characterized by spectroscopic means. Furthermore, molecular structures were determined by single-crystal X-ray diffraction analyses of HL3, 1-3, 9-11 together with three polynuclear products {[MoO(L2)2]2(µ-O)} (7), {[MoO(L2)]4(µ-O)6} (8) and [C9H13N2O]4[Mo8O26]·6OPMe3 (12) which were obtained during the synthesis of reduced complexes of the type [MoO(PMe3)L2] (4-6).

Artifacts in Sonography - Part 3.

As a continuation of parts 1 1 and 2 2, this article discusses artifacts as caused by insufficient temporal resolution, artifacts in color and spectral Doppler sonography, and information regarding artifacts in sonography with contrast agents. There are artifacts that occur in B-mode sonography as well as in Doppler imaging methods and sonography with contrast agents, such as slice thickness artifacts and bow artifacts, shadows, mirroring, and artifacts due to refraction that appear, for example, as double images, because they are based on the same formation mechanisms. In addition, there are artifacts specific to Doppler sonography, such as the twinkling artifact, and method-based motion artifacts, such as aliasing, the ureteric jet, and due to tissue vibration. The artifacts specific to contrast mode include echoes from usually highly reflective structures that are not contrast bubbles ("leakage"). Contrast agent can also change the transmitting signal so that even structures not containing contrast agent are echogenic ("pseudoenhancement"). While artifacts can cause problems regarding differential diagnosis, they can also be useful for determining the diagnosis. Therefore, effective use of sonography requires both profound knowledge and skilled interpretation of artifacts.

Targeted Long-Read Sequencing of a Locus Under Long-Term Balancing Selection in Capsella.

Rapid advances in short-read DNA sequencing technologies have revolutionized population genomic studies, but there are genomic regions where this technology reaches its limits. Limitations mostly arise due to the difficulties in assembly or alignment to genomic regions of high sequence divergence and high repeat content, which are typical characteristics for loci under strong long-term balancing selection. Studying genetic diversity at such loci therefore remains challenging. Here, we investigate the feasibility and error rates associated with targeted long-read sequencing of a locus under balancing selection. For this purpose, we generated bacterial artificial chromosomes (BACs) containing the Brassicaceae S-locus, a region under strong negative frequency-dependent selection which has previously proven difficult to assemble in its entirety using short reads. We sequence S-locus BACs with single-molecule long-read sequencing technology and conduct de novo assembly of these S-locus haplotypes. By comparing repeated assemblies resulting from independent long-read sequencing runs on the same BAC clone we do not detect any structural errors, suggesting that reliable assemblies are generated, but we estimate an indel error rate of 5.7×10-5 A similar error rate was estimated based on comparison of Illumina short-read sequences and BAC assemblies. Our results show that, until de novo assembly of multiple individuals using long-read sequencing becomes feasible, targeted long-read sequencing of loci under balancing selection is a viable option with low error rates for single nucleotide polymorphisms or structural variation. We further find that short-read sequencing is a valuable complement, allowing correction of the relatively high rate of indel errors that result from this approach.

Dye Tool Box for a Fluorescence Enhancement Immunoassay.

Immunochemical analytical methods are very successful in clinical diagnostics and are nowadays also emerging in the control of food as well as monitoring of environmental issues. Among the different immunoassays, luminescence based formats are characterized by their outstanding sensitivity making this format especially attractive for future applications. The need for multiparameter detection capabilities calls for a tool box of dye labels in order to transduce the biochemical reaction into an optically detectable signal. Here, in a multiparameter approach each analyte may be detected by a different dye with a unique emission color (covering the blue to red spectral range) or a unique luminescence decay kinetics. In the case of a competitive immunoassay format for each of the different dye labels an individual antibody would be needed. In the present paper a slightly modified approach is presented using a 7-aminocoumarin unit as the basic antigen against which highly specific antibodies were generated. Leaving the epitope region in the dyes unchanged but introducing a side group in positon 3 of the coumarin system allowed us to tune the optical properties of the coumarin dyes without the necessity of new antibody generation. Upon modification of the parent coumarin unit the full spectral range from blue to deep red was accessed. In the manuscript the photophysical characterization of the coumarin derivatives and their corresponding immunocomplexes with two highly specific antibodies is presented. The coumarin dyes and their immunocomplexes were characterized by steady-state and time-resolved absorption as well as emission spectroscopy. Moreover, fluorescence depolarization measurements were carried out to complement the data stressing the different binding modes of the two antibodies. The binding modes were evaluated using the photophysics of 7-aminocoumarins and how it was affected in the respective immunocomplexes, namely, the formation of the intramolecular charge transfer (ICT) as well as the twisted intramolecular charge transfer (TICT). In contrast to other antibody-dye pairs reported a distinct fluorescence enhancement upon formation of the antibody-dye complex up to a factor of 50 was found. Because of the easy emission color tuning by tailoring the coumarin substitution for the antigen binding in nonrelevant position 3 of the parent molecule, a dye tool box is on hand which can be used in the construction of competitive multiparameter fluorescence enhancement immunoassays (FenIA).

Antibody Binding at the Liposome-Water Interface: A FRET Investigation toward a Liposome-Based Assay.

Different signal amplification strategies to improve the detection sensitivity of immunoassays have been applied which utilize enzymatic reactions, nanomaterials, or liposomes. The latter are very attractive materials for signal amplification because liposomes can be loaded with a large amount of signaling molecules, leading to a high sensitivity. In addition, liposomes can be used as a cell-like "bioscaffold" to directly test recognition schemes aiming at cell-related processes. This study demonstrates an easy and fast approach to link the novel hydrophobic optical probe based on [1,3]dioxolo[4,5-f]-[1,3]benzodioxole (DBD dye mm239) with tunable optical properties to hydrophilic recognition elements (e.g., antibodies) using liposomes for signal amplification and as carrier of the hydrophobic dye. The fluorescence properties of mm239 (e.g., long fluorescence lifetime, large Stokes shift, high photostability, and high quantum yield), its high hydrophobicity for efficient anchoring in liposomes, and a maleimide bioreactive group were applied in a unique combination to build a concept for the coupling of antibodies or other protein markers to liposomes (coupling to membranes can be envisaged). The concept further allowed us to avoid multiple dye labeling of the antibody. Here, anti-TAMRA-antibody (DC7-Ab) was attached to the liposomes. In proof-of-concept, steady-state as well as time-resolved fluorescence measurements (e.g., fluorescence depolarization) in combination with single molecule detection (fluorescence correlation spectroscopy, FCS) were used to analyze the binding interaction between DC7-Ab and liposomes as well as the binding of the antigen rhodamine 6G (R6G) to the antibody. Here, the Förster resonance energy transfer (FRET) between mm239 and R6G was monitored. In addition to ensemble FRET data, single-molecule FRET (PIE-FRET) experiments using pulsed interleaved excitation were used to characterize in detail the binding on a single-molecule level to avoid averaging out effects.

Risk-factors for nodular hyperplasia of parathyroid glands in sHPT patients.

INTRODUCTION: Nodular hyperplasia of parathyroid glands (PG) is the most probable cause of medical treatment failure in secondary hyperparathyroidism (sHPT). This prospective cohort study is located at the interface of medical and surgical consideration of sHPT treatment options and identifies risk-factors for nodular hyperplasia of PG. MATERIAL AND METHODS: One-hundred-eight resected PG of 27 patients with a broad spectrum of sHPT severity were classified according to the degree of hyperplasia by histopathology. Twenty routinely gathered parameters from medical history, ultrasound findings of PG and laboratory results were analyzed for their influence on nodular hyperplasia of PG by risk-adjusted multivariable binary regression. A prognostic model for non-invasive assessment of PG was developed and used to weight the individual impact of identified risk-factors on the probability of nodular hyperplasia of single PG. RESULTS: Independent risk-factors for nodular hyperplasia of single PG were duration of dialysis in years, PG volume in mm3 determined by ultrasound and serum level of parathyroid hormone in pg/mL. Multivariable analyses computed a model with an Area Under the Receiver Operative Curve of 0.857 (95%-CI:0.773-0.941) when predicting nodular hyperplasia of PG. Theoretical assessment of risk-factor interaction revealed that the duration of dialysis had the strongest influence on the probability of nodular hyperplasia of single PG. CONCLUSIONS: The three identified risk-factors (duration of dialysis, PG volume determined by ultrasound and serum level of parathyroid hormone) can be easily gathered in daily routine and could be used to non-invasively assess the probability of nodular hyperplasia of PG. This assessment would benefit from periodically collected data sets of PG changes during the course of sHPT, so that the choice of medical or surgical sHPT treatment could be adjusted more to the naturally changing type of histological PG lesion on an individually adopted basis in the future.

Unusual C-N Coupling Reactivity of Thiopyridazines: Efficient Synthesis of Iron Diorganotrisulfide Complexes.

The reaction of iron(II) triflate with 6-tert-butyl-3-thiopyridazine (PnH) and 4-methyl-6-tert-butyl-3-thiopyridazine (MePnH) respectively led to iron bis(diorganotrisulfide) complexes [Fe(RPnS3PnR)2](OTf)2 [R = H (1a) and Me (2a)]. The corresponding perchlorate complexes were prepared by using the iron(II) chloride precursor and the subsequent addition of 2 equiv of NaClO4, giving [Fe(RPnS3PnR)2](ClO4)2 [R = H (1b) and Me (2b)]. The compounds were fully characterized including single-crystal X-ray diffraction analysis. All four compounds exhibit nearly perfect octahedral geometries with an iron center coordinated by four nitrogen atoms from two RPnS3PnR ligands and by two sulfur atoms of the central atom in the S3 unit. The diamagnetic complexes exhibit unusually high redox potentials for the Fe2+/3+ couple at E1/2 = 1.15 V (for 1a and 1b) and 1.08 V (for 2a and 2b) versus Fc/Fc+, respectively, as determined by cyclic voltammetry. Furthermore, the source of the extra sulfur atom within the S3 unit was elucidated by isolation of C-N-coupled pyridazinylthiopyridazine products.