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To shed light on nanoscale pathologies in patients with polyneuropathy, we assessed human nerve biopsies by super-resolution fluorescence microscopy. We focused on both physiological protein arrangement and pathological ultrastructural changes at the node of Ranvier, a crucial region of the peripheral myelinated axon. Direct stochastic optical reconstruction microscopy (dSTORM) revealed a ∼190 nm periodic protein arrangement of cytoskeletal proteins and axoglial cell adhesion molecules. Periodic distances increased at the paranodal region of the node of Ranvier in patients with polyneuropathy, both at the axonal cytoskeleton and at the axoglial junction. In-depth image analysis of human nerve biopsies revealed a partial loss of proteins of the axoglial complex (Caspr-1, neurofascin-155) in combination with detachment from the cytoskeletal anchor protein ß2-spectrin. Super-resolution dual-color colocalization data was supported by high-content confocal imaging combined with deep learning-based analysis, indicating that paranodal elongation occurs especially in acute and severe axonal neuropathy, as a possible correlate of Wallerian degeneration and related cytoskeletal damage. Our findings show that super-resolution imaging can identify, quantify and map elongated periodic protein distances in peripheral nerve biopsies for pathophysiological studies and direct implications for diagnostic assessment.Graphical abstractPathological alterations of ultrastructural protein arrangement at the paranodal region of the node of Ranvier in polyneuropathy.Super-resolution microscopy allows for assessing ultrastructural protein arrangement and pathological alterations in patients with polyneuropathy. In human healthy nodes, axoglial and axoskeletal proteins follow a 190 nm periodic arrangement (left). In pathologically altered nodes (right), periodic protein distances of axonal ß2-spectrin elongate, in combination with elongation and partial loss of the axoglial complex of Caspr-1 and neurofascin-155. The axoglial complex itself colocalizes closely even in pathologically altered nodes. A complete loss of the axoglial complex could be the ultrastructural correlate of the detachment of paranodal myelin loops. Scale bar 2 μm.

Supplementary figures of custom tools and troubleshooting table., Related Publication

In recent years, the development of new image analysis approaches has highlighted the possibility of recovering superresolution information from short sequences of wide-field images. Our recently developed method, SRRF (Super-Resolution Radial Fluctuations), enables long-term live-cell imaging beyond the resolution limit without specialized hardware. Here, we present eSRRF (enhanced-SRRF), a significant improvement over our initial method, enhancing image fidelity to the underlying structure and resolution. Especially, eSRRF uses automated data-driven parameter optimization, including an estimation of the number of frames necessary for optimal reconstruction. We demonstrate the improved fidelity of the images reconstructed with eSRRF and highlight its versatility and ease of use over a wide range of microscopy techniques and biological systems. We also extend eSRRF to 3D super-resolution microscopy by combining it with multi-focus microscopy (MFM), obtaining volumetric super-resolution imaging of live cells with acquisition speed of ~1 volume/second.

The unicellular eukaryote

Store-operated Ca(2+) entry (SOCE) is the major route of Ca(2+) influx in platelets. The Ca(2+) sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca(2+) channel Orai1 and the inositol trisphosphate receptor (IP(3)R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP(3)R in platelets. Deletion of platelet BIN2 (Bin2(fl/fl,Pf4-Cre) mice) resulted in reduced Ca(2+) store release and Ca(2+) influx in response to all tested platelet agonists. These defects were a consequence of impaired IP(3)R function in combination with defective STIM1-mediated SOC channel activation, while Ca(2+) store content and agonist-induced IP(3) production were unaltered. This severely defective Ca(2+) signaling translated into impaired thrombus formation under flow and a protection of Bin2(fl/fl,Pf4-Cre) mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

Interrogating platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke, or pulmonary embolism. The underlying key receptor distributions and interactions, in particular the relevance of integrin clustering, are not fully understood is because of highly abundant and densely distributed αIIbβ3 receptors. This makes receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Here, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We show that 4x expansion is highly straight-forward for super-resolution microscopy of platelets, while 10x expansion provides higher precision at the price of increased efforts in sample preparation and imaging. Quantifying various receptor colocalization scenarios we demonstrate that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. We reveal the presence of αIIbβ3 clusters in resting platelets, as well as in activated platelets, indicating that they contribute to the rapid platelet response during platelet clotting.

Understanding the structure of supramolecular complexes provides insight into their functional capabilities and how they can be modulated in the context of disease. Super-resolution microscopy (SRM) excels in performing this task by resolving ultrastructural details at the nanoscale with molecular specificity. However, technical limitations, such as underlabelling, preclude its ability to provide complete structures. Single-particle analysis (SPA) overcomes this limitation by combining information from multiple images of identical structures and producing an averaged model, effectively enhancing the resolution and coverage of image reconstructions. This review highlights important studies using SRM-SPA, demonstrating how it broadens our knowledge by elucidating features of key biological structures with unprecedented detail., Comment: 12 pages, 3 figures

Neutron-induced reaction cross sections of unstable nuclei are essential for understanding the synthesis of heavy elements in stars and for applications in nuclear technology. However, their measurement is very complicated due to the radioactivity of the targets involved. We propose to circumvent this problem by using the surrogate reaction method in inverse kinematics, where the nucleus formed in the neutron- induced reaction of interest is produced by a reaction involving a radioactive heavy-ion beam and a stable, light target nucleus. The probabilities as a function of the compound-nucleus excitation energy for γ-ray emission, neutron emission and fission, which can be measured with the surrogate reaction, are particularly useful to constrain model parameters and to obtain more accurate predictions of the neutron-induced reaction cross sections of interest. Yet, the full development of the surrogate method is hampered by numerous long- standing target issues, which can be solved by combining surrogate reactions with the unique and largely unexplored possibilities at heavy-ion storage rings. In this contribution, we describe the developments we are carrying out to measure for the first time simultaneously γ-ray emission, neutron emission and fission probabilities at the storage rings of the GSI/FAIR facility. In particular, we will present the first results of the proof of principle experiment, which we performed in June 2022 at the Experimental Storage Ring (ESR) of GSI/FAIR.

BackgroundOptimal dosing of methotrexate (MTX) in rheumatoid arthritis (RA) remains challenging. To this end, monitoring of intracellular MTX polyglutamates (MTX-PGs) in red blood cells (RBCs) has been investigated as a potential marker of MTX (non-)response, with contradictory results. As enucleated, non-proliferative cells, mature RBCs lack regulated folate metabolism and are devoid of folylpolyglutamate synthetase (FPGS) activity catalyzing the conversion of MTX to MTX-PGs. Therefore, it has been argued that analysis of MTX-PG in immune-effector cells, represented by peripheral blood mononuclear cells (PBMCs), would be more relevant. However, no prospective study has been performed measuring MTX-PG levels in PBMCs nor in comparison with RBCs.ObjectivesTo investigate the pharmacokinetics of MTX-PG accumulation in RBCs and PBMCs in newly diagnosed RA patients in the early phase of MTX treatment.MethodsIn a clinical prospective cohort study (Methotrexate Monitoring (NTR7149)), RA patients were administered MTX op to 25 mg/week, as described before. (1) At 1, 2, 3 and 6 months after start of therapy, blood was collected and RBCs were isolated by centrifugation and PBMCs after Ficoll density gradient centrifugation. MTX-PG1-6 concentrations in these cells were analyzed using a UPLC-MS/MS method with including custom-made stable isotopes of MTX-PG1-6 as internal standards. (2) UPLC-MS/MS measurements for MTX-PG1-6 were performed with a Waters Acquity BEH C18 column coupled to an AB Sciex 6500+ with the ESI operating on the positive mode. MTX dosing and concomitant treatments were in conformity with clinical practice. (3)Results46 consecutive patients were included in this study; 76% female, mean age: 57.8 years, mean baseline DAS28-ESR: 3.5, as described before. (1) Mean dosage was 10.5 mg (SD: 1.5) at baseline, 16.3 mg (2.5) at month 1, 22.7 mg (4.5) at month 2, 19.5 mg (6.3) at month 3 and 19.1 mg (6.2) at month 6.MTX-PG accumulation in PBMCs and RBCs revealed a disparate profile in both MTX-PG distribution and absolute accumulation levels (Figure 1A/B). Remarkably, MTX-PG distribution in PBMCs was mainly composed of MTX-PG1 (58%), and to a lesser extent MTX-PG2 (27%) and MTX-PG3 (15%). Longer chain MTX-PG4-6 were also detectable in PBMCs, but at lower levels (mean: 4.0 – 6.7 fmol/10^6 cells) than MTX-PG1-3. Moreover, this MTX-PG distribution profile in PBMCs remained constant over a MTX therapy period of 6 months (Figure 1A). The RBC MTX-PG accumulation profile shows mainly MTX-PG1 and lower levels of MTX-PG2-6 at 1 month after the start of therapy. After 3 months of therapy, MTX-PG3 is the main PG-moiety with also MTX-PG4,5,6 being detected. This profile is largely similar after 6 months of therapy. With respect to total intracellular MTX-PG1-6 accumulation, PBMCs had significantly (p1-6 levels in RBCs and PBMCs at all time points were weakly correlated (r=0.41, pFigure 1.Individual MTX-PG concentrations in PBMCs (A) and RBCs (B) during the first 6 months of MTX administration (note the different scaling of the y-axes). At 6 months, 36 patients were still on MTX treatment. Panel (C): Spearman’s correlation plot of total MTX-PGs in RBCs versus PBMCs of all time points.The disparate MTX-PG accumulation and distribution profiles in PBMCs versus RBCs of RA patients may be associated with the shorter life span of PBMCs and the low FPGS activity in RBCs. (4)No significant relation between MTX-PGs and DAS28 was found (data not shown).ConclusionThis study shows that in newly diagnosed RA patients starting MTX therapy, MTX-PG concentrations in PBMCs are significantly 10-20-fold higher than in RBCs over a period of 6 months, with a disparate MTX-PG distribution profile in PBMCs (highest: MTX-PG1) than RBCs (highest: MTX-PG3).References[1]RCF Hebing, Arthr Rheum (2021)[2]E den Boer, Anal Bioanal Chem (2013)[3]J Smolen, Ann Rheum Dis (2020)[4]IB Muller, Ther Drug Monit (2019)AcknowledgementsAcknowledgements: We would like to thank all participating patients and Pfizer (grant 53233663 / WI230458), AmsterdamUMC (AI&II extension grant) and NVKC (Noyons grant 2018)Disclosure of InterestsRenske Hebing Grant/research support from: Pfizer (grant 53233663 / WI230458), NVKC (Netherlands Society for Clinical Chemistry, Noyons grant 2018) and AmsterdamUMC (extension grant), Marry Lin: None declared, Eduard Struys: None declared, Sohaila Mahmoud: None declared, Ittai Muller: None declared, Sandra Heil: None declared, Pieter Griffioen: None declared, WIllem Lems: None declared, Bart van den Bemt Speakers bureau: Pfizer, UCB, Sanofi-Aventis, Galapagos, Amgen and Eli Lilly, Michael Nurmohamed Grant/research support from: Pfizer grant 53233663 / WI230458, Gerrit Jansen: None declared, Robert De Jonge Grant/research support from: NVKC (Netherlands Society for Clinical Chemistry, Noyons grant)

The maker movement has reached the optics labs, empowering researchers to create and modify microscope designs and imaging accessories. 3D printing has a disruptive impact on the field, improving accessibility to fabrication technologies in additive manufacturing. This approach is particularly useful for rapid, low-cost prototyping, allowing unprecedented levels of productivity and accessibility. From inexpensive microscopes for education such as the FlyPi to the highly complex robotic microscope OpenFlexure, 3D printing is paving the way for the democratization of technology, promoting collaborative environments between researchers, as 3D designs are easily shared. This holds the unique possibility of extending the open-access concept from knowledge to technology, allowing researchers everywhere to use and extend model structures. Here, it is presented a review of additive manufacturing applications in optical microscopy for life sciences, guiding the user through this new and exciting technology and providing a starting point to anyone willing to employ this versatile and powerful new tool.

DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA., Graphical Abstract Graphical AbstractNGS methylation analysis with pool of randomized CpG double site substrates and SFM imaging of enzyme-DNA complexes with example substrates revealed novel interaction modes of DNMT3A and DNMT3A/3L with CpG double site substrates.

Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tuned mirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor of two. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.

The maker movement has reached the optics labs, empowering researchers to actively create and modify microscope designs and imaging accessories. 3D printing has especially had a disruptive impact on the field, as it entails an accessible new approach in fabrication technologies, namely additive manufacturing, making prototyping in the lab available at low cost. Examples of this trend are taking advantage of the easy availability of 3D printing technology. For example, inexpensive microscopes for education have been designed, such as the FlyPi. Also, the highly complex robotic microscope OpenFlexure represents a clear desire for the democratisation of this technology. 3D printing facilitates new and powerful approaches to science and promotes collaboration between researchers, as 3D designs are easily shared. This holds the unique possibility to extend the open-access concept from knowledge to technology, allowing researchers from everywhere to use and extend model structures. Here we present a review of additive manufacturing applications in microscopy, guiding the user through this new and exciting technology and providing a starting point to anyone willing to employ this versatile and powerful new tool.

Interrogating small platelets and their densely packed, highly abundant receptor landscape is key to understand platelet clotting, a process that can save lives when stopping blood loss after an injury, but also kill when causing heart attack, stroke or pulmonary embolism. The underlying key receptor distributions and interactions, in particular the relevance of integrin clustering, are not fully understood is because of highly abundant and densely distributed GPIIb/IIIa receptors. This makes receptor distributions difficult to assess even by super-resolution fluorescence microscopy. Here, we combine dual-color expansion and confocal microscopy with colocalization analysis to assess platelet receptor organization without the need of a super-resolution microscope. We show that 4x expansion is highly straight-forward for super-resolution microscopy of platelets, while 10x expansion provides higher precision at the price of increased efforts in sample preparation and imaging. Quantifying various receptor colocalization scenarios we demonstrate that expansion microscopy can pinpoint receptor distributions and interactions in resting and activated platelets being superior to conventional methods that fail in such dense 3D scenarios with highly abundant receptors. We reveal the presence of GPIIb/IIIa clusters in resting platelets, which are not affected by platelet activation indicating that they contribute to the rapid platelet response during platelet clotting.Graphical Abstract

Nonsense mutations change an amino acid codon to a premature termination codon (PTC) generally through a single‐nucleotide substitution. The generation of a PTC results in a defective truncated protein and often in severe forms of disease. Because of the exceedingly high prevalence of nonsense‐associated diseases and a unifying mechanism, there has been a concerted effort to identify PTC therapeutics. Most clinical trials for PTC therapeutics have been conducted with small molecules that promote PTC read through and incorporation of a near‐cognate amino acid. However, there is a need for PTC suppression agents that recode PTCs with the correct amino acid while being applicable to PTC mutations in many different genomic landscapes. With these characteristics, a single therapeutic will be able to treat several disease‐causing PTCs. In this review, we will focus on the use of nonsense suppression technologies, in particular, suppressor tRNAs (sup‐tRNAs), as possible therapeutics for correcting PTCs. Sup‐tRNAs have many attractive qualities as possible therapeutic agents although there are knowledge gaps on their function in mammalian cells and technical hurdles that need to be overcome before their promise is realized. This article is categorized under:RNA Processing > tRNA ProcessingTranslation > Translation Regulation, Nonsense suppressor tRNAs display promise as therapeutic agents for suppressing premature termination codons, and alleviating disease through seamless rescue of full‐length protein and stabilized mRNA.

Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding and partly impossible owing to the lack of specific functions or substrates in the host cell, its sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we demonstrate that cell-free systems can be useful in driving the characterization and engineering of biosynthetic pathways. We show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. We prove the concept of this approach on the direct synthesis of indigoidine and rhabdopeptides with the in vitro produced multidomain megasynthases BpsA and KJ12ABC. The in vitro produced proteins are analyzed in detail, i.e., in yield, quality, post-translational modification and specific activity, and compared to recombinantly produced proteins. Our study highlights cell-free PURE systems as suitable setting for the rapid engineering of biosynthetic pathways.

Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions.

Amber codon suppression is a powerful tool to site-specifically modify proteins to generate novel biophysical probes. Yet, its application on large and complex multidomain proteins is challenging, leading to difficulties during structural and conformational characterization using spectroscopic methods. The animal fatty acid synthase type I is a 540 kDa homodimer displaying large conformational variability. As the key enzyme ofde novofatty acid synthesis, it attracts interest in the fields of obesity, diabetes and cancer treatment. Substrates and intermediates remain covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic domains by the acyl carrier protein domain. Thus, conformational variability of animal FAS is an essential aspect for fatty acid biosynthesis. We investigate this multidomain protein as a model system for probing amber codon suppression by genetic encoding of non-canonical amino acids. The systematic approach relies on a microplate-based reporter assay of low complexity, that was used for quick screening of suppression conditions. Furthermore, the applicability of the reporter assay is demonstrated by successful upscaling to both full-length constructs and increased expression scale. The obtained fluorescent probes of murine FAS type I could be subjected readily to a conformational analysis using single-molecule fluorescence resonance energy transfer.

In the vicinity of metallic nanostructures, absorption and emission rates of optical emitters can be modulated by several orders of magnitude1,2. Control of such near-field light–matter interaction is essential for applications in biosensing3, light harvesting4 and quantum communication5,6 and requires precise mapping of optical near-field interactions, for which single-emitter probes are promising candidates7–11. However, currently available techniques are limited in terms of throughput, resolution and/or non-invasiveness. Here, we present an approach for the parallel mapping of optical near-field interactions with a resolution of

The G-protein-coupled receptor (GPCR) superfamily mediates cellular responses and communication across cellular membranes and is the largest known class of molecular targets with proven therapeutic value. For probing conformational changes of GPCRs and others in a live cell setting, fluorescence resonance energy transfer (FRET) is usually the method of choice. FRET probes often require careful labeling procedures, elaborate characterization, and assay optimization to provide both physiologically relevant probes with unaltered pharmacology and a sufficient dynamic range of the FRET changes. Here, we present an approach to optimize the energy transfer without changing the design of the FRET probe. We show that gold-coated glass coverslips reinforce the otherwise forbidden donor–acceptor energy transfer by virtual optimization of the dipole orientation. First, we confirm the resulting enhanced FRET efficiency on our nanocoatings for the inactive M1 muscarinic acetylcholine receptor (mAChR) labeled with a FRE...

De novo biosynthesis of fatty acids is an iterative process requiring strict regulation of the lengths of the produced fatty acids. In this review, we focus on the factors determining chain lengths in fatty acid biosynthesis. In a nutshell, the process of chain-length regulation can be understood as the output of a chain-elongating C-C bond forming reaction competing with a terminating fatty acid release function. At the end of each cycle in the iterative process, the synthesizing enzymes need to "decide" whether the growing chain is to be elongated through another cycle or released as the "mature" fatty acid. Recent research has shed light on the factors determining fatty acid chain length and has also achieved control over chain length for the production of the technologically interesting short-chain (C4 -C8 ) and medium-chain (C10 -C14 ) fatty acids.

Lifetime measurements of excited states in the neutron-rich nucleus ^{43}S were performed by applying the recoil-distance method on fast rare-isotope beams in conjunction with the Gamma-Ray Energy Tracking In-beam Nuclear Array. The new data based on γγ coincidences and lifetime measurements resolve a doublet of (3/2^{-}) and (5/2^{-}) states at low excitation energies. Results were compared to the π(sd)-ν(pf) shell model and antisymmetrized molecular dynamics calculations. The consistency with the theoretical calculations identifies a possible appearance of three coexisting bands near the ground state of ^{43}S: the K^{π}=1/2^{-} band built on a prolate-deformed ground state, a band built on an isomer with a 1f_{7/2}^{-1} character, and a suggested excited band built on a newly discovered doublet state. The latter further confirms the collapse of the N=28 shell closure in the neutron-rich region.

We examined the associations of Epstein-Barr virus (EBV) status with characteristics and outcomes of posttransplantation lymphoproliferative disorder (PTLD) by studying 176 adult solid organ transplant recipients diagnosed with PTLD between 1990 and 2013 (58 [33%] EBV-negative; 118 [67%] EBV-positive). The proportion of EBV-negative cases increased over time from 10% (1990-1995) to 48% (2008-2013) (p < 0.001). EBV-negative PTLD had distinct characteristics (monomorphic histology, longer latency) though high-risk features (advanced stage, older age, high lactate dehydrogenase, central nervous system involvement) were not more common compared to EBV-positive PTLD. In multivariable analysis, EBV negativity was not significantly associated with worse response to initial therapy (adjusted odds ratio, 0.84; p = 0.75). The likelihood of achieving a complete remission (CR) was not significantly different for EBV-negative versus EBV-positive PTLD including when therapy was reduction of immunosuppression alone (35% vs. 43%, respectively, p = 0.60) or rituximab (43% vs. 47%, p = 1.0). EBV negativity was also not associated with worse overall survival (adjusted hazard ratio, 0.91; p = 0.71). Our findings indicate that EBV status is not prognostic or predictive of treatment response in adults with PTLD. The high proportion of EBV-negative disease diagnosed in recent years highlights the need for new strategies for prevention and management of EBV-negative PTLD.

Super-resolution techniques such as direct Stochastic Optical Reconstruction Microscopy (dSTORM) have become versatile and well-established tools for biological imaging over the last century. Here, we theoretically and experimentally show that clever combination of different fluorescence modalities allows further improvements. We found that the interaction of fluorophores with plasmonic surfaces boost super-resolution performance in dSTORM approaches as it allows for tailoring the excitation and emission properties. The strength of the approach is that no further specialized microscope setup is required as the described enhancement solely rely on metal-dielectric coated glass coverslips that are straightforward to fabricate. Such biocompatible plasmonic nanolayers enhance the signal-to-noise ratio of dSTORM, and thus sharpens the localization precision by a factor of two.

Background and purpose Primary MRI diagnosis of spinal intramedullary tumor-suspected lesions can be challenging and often requires spinal biopsy or resection with a substantial risk of neurological deficits. We evaluated whether Diffusion Tensor Imaging (DTI) tractography can facilitate the differential diagnosis. Materials and methods Twenty-five consecutive patients with an intramedullary tumor-suspected lesion considered for spinal surgery were studied with a Diffusion-weighted multi-shot read out segmented EPI sequence (RESOLVE). White matter tracts (“streamlines”) were calculated using the FACT algorithm and visually co-registered to a T2-weighted 3D sequence. The fused images were assessed concerning spinal streamline appearance as normal, displaced or terminated. Definite diagnosis was verified by histological analysis or further clinical work-up. Results All patients with normal appearing streamlines (n = 6) showed an acute inflammatory demyelinating pathology in the further clinical work-up. In 10 patients streamline displacing lesions were found from which 5 patients underwent a surgical treatment with histologically confirmed low-grade tumors like ependymomas and pilocytic astrocytomas. In nine patients streamlines were terminated, from which 6 patients received a histology proven diagnoses with a more heterogenous spectrum (3 cases of high grade tumor, 1 case of low grade tumor with intralesional hemorrhage and 2 cases with gliosis but no tumor cells). Conclusion Using multi-shot DTI spinal tractography acute inflammatory lesions can be differentiated from other tumorous intramedullary lesions. The entity diagnosis of spinal tumors seems to be more challenging, primarily due to the variety of factors like invasivity, expansion or intralesional hemorrhage., BACKGROUND AND PURPOSE: Primary MRI diagnosis of spinal intramedullary tumor-suspected lesions can be challenging and often requires spinal biopsy or resection with a substantial risk of neurological deficits. We evaluated whether Diffusion Tensor Imaging (DTI) tractography can facilitate the differential diagnosis. MATERIALS AND METHODS: Twenty-five consecutive patients with an intramedullary tumor-suspected lesion considered for spinal surgery were studied with a Diffusion-weighted multi-shot read out segmented EPI sequence (RESOLVE). White matter tracts ("streamlines") were calculated using the FACT algorithm and visually co-registered to a T2-weighted 3D sequence. The fused images were assessed concerning spinal streamline appearance as normal, displaced or terminated. Definite diagnosis was verified by histological analysis or further clinical work-up. RESULTS: All patients with normal appearing streamlines (n=6) showed an acute inflammatory demyelinating pathology in the further clinical work-up. In 10 patients streamline displacing lesions were found from which 5 patients underwent a surgical treatment with histologically confirmed low-grade tumors like ependymomas and pilocytic astrocytomas. In nine patients streamlines were terminated, from which 6 patients received a histology proven diagnoses with a more heterogenous spectrum (3 cases of high grade tumor, 1 case of low grade tumor with intralesional hemorrhage and 2 cases with gliosis but no tumor cells). CONCLUSION: Using multi-shot DTI spinal tractography acute inflammatory lesions can be differentiated from other tumorous intramedullary lesions. The entity diagnosis of spinal tumors seems to be more challenging, primarily due to the variety of factors like invasivity, expansion or intralesional hemorrhage.

Purpose The relative merits of various forms of antidepressant therapy in patients with heart failure (HF) are discussed, including disease-specific pharmacokinetic changes and drug-interaction challenges in current or likely future candidates for heart transplantation. Summary There is a growing emphasis on the use of antidepressants in patients with chronic HF, as depression can have a negative impact on HF progression and morbidity and mortality after heart transplants or other invasive cardiac surgery. Evidence from one small study of patients with concomitant end-stage HF and major depression indicated a reduced risk of cardiovascular death in those receiving β-blockers in combination with selective serotonin-reuptake inhibitor (SSRI) therapy. In addition to pharmacokinetic changes caused by HF itself, which can decrease medication absorption and distribution, other issues to consider in the drug selection process include the potential for antidepressants to interact with posttransplant immunosuppressive therapy and the possible effects of antidepressant use on surgical transfusion requirements. The SSRIs are generally recommended as first-line therapies for depressed patients with HF; however, fluvoxamine and fluoxetine should be avoided due to interactions with immunosuppressant agents. If SSRI therapy is not well tolerated or adjunctive therapy is required, bupropion, mirtazapine, venlafaxine, and duloxetine may be suitable alternatives for certain patients. Conclusion Key considerations in antidepressant selection in the context of advanced HF include HF-related changes in drug pharmacokinetics that may affect initial dosing or dosage adjustment, possible drug interactions, adverse effects that may potentiate those induced by immunosuppressants added after transplantation, and tolerability issues.

In the vicinity of metallic nanostructures, absorption and emission rates of optical emitters can be modulated by several orders of magnitude

Venous thromboembolism (VTE) is a common but often overlooked life-threatening complication of critical illness. The aim of this cross-sectional survey was to assess current practice of thromboprophylaxis as well as adherence to international guidelines.After ethics committee approval, all intensive care units in Austrian hospitals treating adult patients were invited to participate in this web-based survey. Anonymized data on each patient treated at the participating intensive care units on Coagulation Day 2010 were collected using an electronic case report form. Risk assessment, choice and monitoring of anticoagulants, means of mechanical prophylaxis, and demographic data were recorded.Data from 325 critically ill patients were collected. Patients had a median of four risk factors for thrombosis and 6 % suffered from VTE. Of the 325 patients, 80 % received low molecular weight heparins subcutaneously, 10 % received unfractionated heparin intravenously, 1 % received alternative anticoagulants and 9 % received no pharmacological prophylaxis. Mechanical prophylaxis was used in 49 % with a predominant use of graduated compression stockings. In 39 % a combination of pharmacological and mechanical prophylaxis was applied and 5 % received no prophylaxis at all. Overall guideline adherence was 40 % on Coagulation Day 2010.Current practice of thromboprophylaxis is predominantly based on the administration of low molecular weight heparins prescribed at rather arbitrary doses without a discernible relationship to drug monitoring, thromboembolic risk factors, vasopressor use or fluid balance. The use of mechanical prophylaxis, evaluation of risk scores and overall guideline adherence must be further encouraged by education, training and communication.

Fluorescence microscopy allows specific and selective imaging of biological samples. Unfortunately, unspecific background due to auto-fluorescence, scattering, and non-ideal labeling efficiency often adversely affect imaging. Surface plasmon-coupled emission (SPCE) is known to selectively mediate fluorescence that spatially originates from regions close to the metal interface. However, SPCE combined with fluorescence imaging has not been widely successful so far, most likely due to its limited photon yield, which makes it tedious to identify the exact window of the application. As the strength of SPCE based imaging is its unique sectioning capabilities. We decided to identify its clear beneficial operational regime for biological settings by interrogating samples in the presence of ascending background levels. For fluorescent beads as well as live-cell imaging as examples, we show how to extend the imaging performance in extremely high photon background environments. In a common setup using plasmonic gold-coated coverslips using an objective-based total internal reflection fluorescence microscope (TIRF-M), we theoretically and experimentally characterize our fluoplasmonics (f-Pics) approach by providing general user guidance in choosing f-Pics over TIRF-M or classical wide-field (WF).

In this work the choice of appropriate test structures and characterization methods for recessed Ohmic contacts to AlGaN/GaN is discussed. It is shown that, in the worst-case scenario, the prevailing assumption of identical sheet resistance between and under the contacts can lead to errors of up to 3000 % in the extracted specific contact resistance.

Objective Mutations of the SCN9A gene can cause ion channel dependant pain syndromes, generalized epilepsy with febrile seizures or familial febrile seizures, following an autosomal dominant or recessive inheritance. Furthermore cardiac symptoms such as bradycardia with syncopes up to asystole and tachycardia have been described. We present a case of a familial generalized epilepsy with additional paroxysmal tachycardia that has successfully been treated with carbamazepine. Case description Our 5-year-old patient first had absence-like seizures at the age of 3 years, with a frequency of approximately twice a month and a duration of 5 to 10 seconds. The EEG showed paroxysmal generalized polyspike wave complexes. Since the age of 4 years the patient had also suffered from episodes with paroxysmal tachycardia. The patient's mother and her sister had similar seizures in early childhood, both were successfully treated with carbamazepine (CBZ). The mother also suffers from episodes with paroxysmal tachycardia which are accompanied by panic attacks. In our patient, the heterozygous modification c.684C>G; p.I228M was detected in the SCN9A gene, using “next generation sequencing”. The mutation could also be detected in the patient's mother. A therapy with CBZ reduced both the boy's seizure frequency and the frequency of his paroxysmal tachycardia significantly. Discussion SCN9A mutations have also been described for Dravet syndrome and familial febrile seizures, whereas our patient showed a familial absence-like generalized epilepsy in combination with paroxysmal tachycardia. Our patient, his mother and also his aunt were successfully treated with CBZ, which could suggest that the patient's c.684C>G; p.I228M mutation causes a “gain of function” of the Nav1.7 sodium channel. The additional cardiac symptoms could also be an effect of the SCN9A mutation.

The purpose of this paper is to show that neural networks may be promising tools for data compression without loss of information. We combine predictive neural nets and statistical coding techniques to compress text files. We apply our methods to certain short newspaper articles and obtain compression ratios exceeding those of the widely used Lempel-Ziv algorithms (which build the basis of the UNIX functions "compress" and "gzip"). The main disadvantage of our methods is that they are about three orders of magnitude slower than standard methods.

Heparin-induced thrombocytopenia (HIT) type II is a life-threatening complication of heparin therapy. The present case report describes the therapeutic management of HIT type II with thrombosis using the direct thrombin inhibitor argatroban in an intensive care patient after successful surgery of a ruptured infrarenal abdominal aortic aneurysm. Despite high dosing and long-term application of argatroban, anticoagulation remained uncritical and was well controllable by monitoring the activated partial thromboplastin time. In consideration of the pharmacological characteristics, therapy suspension due to invasive interventions and switching to an oral vitamin K antagonist by defined algorithm resulted in an effective management.

A method for pesticide residue analysis applying the technique of two-dimensional gas chromatography with effluent splitting to three selective detectors in parallel is described. Screening and confirmation procedures are combined in one single instrument. The column combination used allows the separation and identification of more than three hundred pesticides on the basis of the resulting retention times and detector signals. Critical pairs of pesticides are distinguished by using the response ratio of the selective detectors characteristic for each substance. An example is given to demonstrate the merits of the described GC-system using the multimethod S19 of the DFG Pesticide Commission for clean-up.

8578 Background: Post-transplant lymphoproliferative disorder (PTLD) is a potentially life-threatening complication of solid organ transplantation. PTLD is frequently linked with Epstein-Barr virus (EBV) and it was suggested that EBV negativity is associated with a poor prognosis and lack of response to reduction of immunosuppression (RI). We conducted a case-control study to identify the characteristics, outcome and response to therapy of EBVpos and EBVneg PTLD over a 20-year period. Methods: We reviewed data on patients diagnosed with PTLD at U. Penn. between 1982 and 2012. We determined EBV positivity on tumor samples according to WHO criteria. We compared clinical and pathologic characteristics, response to therapy, and survival of EBVpos and EBVneg patients. Results: Of 222 patients diagnosed with PTLD, we verified the EBV status of 169 patients, of whom 35% were EBVneg and 65% were EBVpos. Mean follow-up was 46.7 months. Median time from transplant to PTLD was 23.1 mo. in EBVpos vs. 59.3 mo. in EBVneg (p=0.003) with 42% of EBVpos patients being diagnosed within the first year after transplant vs. 15% in the EBVneg group (p