K+ Efflux-Independent NLRP3 Inflammasome Activation by Small Molecules Targeting Mitochondria.

Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.

Bidirectional sequestration between a bacterial hibernation factor and a glutamate metabolizing protein.

Bacterial hibernating 100S ribosomes (the 70S dimers) are excluded from translation and are protected from ribonucleolytic degradation, thereby promoting long-term viability and increased regrowth. No extraribosomal target of any hibernation factor has been reported. Here, we discovered a previously unrecognized binding partner (YwlG) of hibernation-promoting factor (HPF) in the human pathogen Staphylococcus aureus. YwlG is an uncharacterized virulence factor in S. aureus. We show that the HPF-YwlG interaction is direct, independent of ribosome binding, and functionally linked to cold adaptation and glucose metabolism. Consistent with the distant resemblance of YwlG to the hexameric structures of nicotinamide adenine dinucleotide (NAD)-specific glutamate dehydrogenases (GDHs), YwlG overexpression can compensate for a loss of cellular GDH activity. The reduced abundance of 100S complexes and the suppression of YwlG-dependent GDH activity provide evidence for a two-way sequestration between YwlG and HPF. These findings reveal an unexpected layer of regulation linking the biogenesis of 100S ribosomes to glutamate metabolism.

Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters.

Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs are able to incorporate different lanthanides (Lns) in their active site. Dye-coupled assays showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Despite widespread use, there are limitations: oftentimes a pH of 9 and activators are required for the assay. Moreover, Ln-MDH variants are not obtained by isolation from the cells grown with the respective Ln, but by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDHs and the assessment of the enzyme activity using the dye-coupled assay. We compare these results with a protein-coupled assay using its physiological electron acceptor cytochrome cGJ (cyt cGJ ). Depending on the assay, two distinct trends are observed among the Ln series. The specific enzyme activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds that measured by the dye-coupled assay. This suggests that early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

Unveiling the Dynamic Self-Assembly of a Recombinant Dragline-Silk-Mimicking Protein.

Despite the considerable interest in the recombinant production of synthetic spider silk fibers that possess mechanical properties similar to those of native spider silks, such as the cost-effectiveness, tunability, and scalability realization, is still lacking. To address this long-standing challenge, we have constructed an artificial spider silk gene using Golden Gate assembly for the recombinant bacterial production of dragline-mimicking silk, incorporating all the essential components: the N-terminal domain, a 33-residue-long major-ampullate-spidroin-inspired segment repeated 16 times, and the C-terminal domain (N16C). This designed silk-like protein was successfully expressed in Escherichia coli, purified, and cast into films from formic acid. We produced uniformly 13C-15N-labeled N16C films and employed solid-state magic-angle spinning nuclear magnetic resonance (NMR) for characterization. Thus, we could demonstrate that our bioengineered silk-like protein self-assembles into a film where, when hydrated, the solvent-exposed layer of the rigid, ß-nanocrystalline polyalanine core undergoes a transition to an α-helical structure, gaining mobility to the extent that it fully dissolves in water and transforms into a highly dynamic random coil. This hydration-induced behavior induces chain dynamics in the glycine-rich amorphous soft segments on the microsecond time scale, contributing to the elasticity of the solid material. Our findings not only reveal the presence of structurally and dynamically distinct segments within the film's superstructure but also highlight the complexity of the self-organization responsible for the exceptional mechanical properties observed in proteins that mimic dragline silk.

SERPINC1 c.1247dupC: a novel SERPINC1 gene mutation associated with familial thrombosis results in a secretion defect and quantitative antithrombin deficiency.

BACKGROUND: Antithrombin (AT) is an important anticoagulant in hemostasis. We describe here the characterization of a novel AT mutation associated with clinically relevant thrombosis. A pair of sisters with confirmed type I AT protein deficiency was genetically analyzed on suspicion of an inherited SERPINC1 mutation. A frameshift mutation, c.1247dupC, was identified and the effect of this mutation was examined on the cellular and molecular level. METHODS: Plasmids for the expression of wild-type (WT) and mutated SERPINC1 coding sequence (CDS) fused to green fluorescent protein (GFP) or hemagglutinin (HA) tag were transfected into HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot. RESULTS: The c.1247dupC mutation results in a frameshift in the CDS of the SERPINC1 gene and a subsequently altered amino acid sequence (p.Ser417LysfsTer48). This alteration affects the C-terminus of the AT antigen and results in impaired secretion as confirmed by GFP- and HA-tagged mutant AT analyzed in HEK293T cells. CONCLUSION: The p.Ser417LysfsTer48 mutation leads to impaired secretion, thus resulting in a quantitative AT deficiency. This is in line with the type I AT deficiency observed in the patients.

The Protein S Erlangen Mutation PROS1c.1904T>C (F635S) Suppresses Secretion.

BACKGROUND: The recently identified PROS1 mutation Protein S Erlangen c.1904T>C, resulting in amino acid exchange F635S, is associated with severe quantitative protein S (PS) deficiency and clinical thrombosis. It was hypothesized that this deficiency is due to a secretion defect [1]. This report aims to further elucidate the potential secretion defect of PS Erlangen. METHODS: Coding sequences (CDS) of wild type (WT) PROS1 (encoding PS) and mutated PROS1c.1904T>C (encoding PSF635S) were cloned in front of the CDS of green fluorescent protein (GFP), and the respective plasmids were introduced into HEK293T cells. PROS1-GFP and PROS1c.1904T>C-GFP expressing HEK293T cell lines were analyzed by confocal laser scanning microscopy and western blot for cellular proteins and proteins secreted to the growth medium. RESULTS: Western blot analysis revealed a significantly reduced secretion of PSF635S compared to WT PS. This observation was confirmed by the detection of mutant PSF635S-GFP fusion exclusively in the endoplasmic reticulum (ER), while PS-GFP passed through the entire secretory pathway, as indicated by the localization within both the ER and Golgi apparatus. CONCLUSIONS: The Protein S Erlangen mutation results in type I PS deficiency caused by a secretion defect.

Efficient Tandem Copper-Catalyzed Click Synthesis of Multisugar-Modified Oligonucleotides.

Nucleic acids in the form of siRNA, antisense oligonucleotides or mRNA are currently explored as new promising modalities in the pharmaceutical industry. Particularly, the success of mRNA-vaccines against SARS-CoV-2, along with the successful development of the first sugar-modified siRNA therapeutics has inspired the field. The development of nucleic acid therapeutics requires efficient chemistry to link oligonucleotides to chemical structures that can improve stability, boost cellular uptake, or enable specific targeting. For the siRNA therapeutics currently in use, modification of the 3'-end of the oligonucleotides with triple-N-acetylgalactosamine (GalNAc)3 was shown to be of significance. This modification is currently achieved through cumbersome multistep synthesis and subsequent loading onto the solid support material. Herein, we report the development of a bifunctional click-reactive linker that allows the modification of oligonucleotides in a tandem click reaction with multiple sugars, regardless of the position within the oligonucleotide, with remarkable efficiency and in a one-pot reaction.

Unexpected Roles for the Second Brain: Enteric Nervous System as Master Regulator of Bowel Function.

At the most fundamental level, the bowel facilitates absorption of small molecules, regulates fluid and electrolyte flux, and eliminates waste. To successfully coordinate this complex array of functions, the bowel relies on the enteric nervous system (ENS), an intricate network of more than 500 million neurons and supporting glia that are organized into distinct layers or plexi within the bowel wall. Neuron and glial diversity, as well as neurotransmitter and receptor expression in the ENS, resembles that of the central nervous system. The most carefully studied ENS functions include control of bowel motility, epithelial secretion, and blood flow, but the ENS also interacts with enteroendocrine cells, influences epithelial proliferation and repair, modulates the intestinal immune system, and mediates extrinsic nerve input. Here, we review the many different cell types that communicate with the ENS, integrating data about ENS function into a broader view of human health and disease. In particular, we focus on exciting new literature highlighting relationships between the ENS and its lesser-known interacting partners.

An Exploratory Study Using Next-Generation Sequencing to Identify Prothrombotic Variants in Patients with Cerebral Vein Thrombosis.

Prothrombotic hereditary risk factors for cerebral vein thrombosis (CVT) are of clinical interest to better understand the underlying pathophysiology and stratify patients for the risk of recurrence. This study explores prothrombotic risk factors in CVT patients. An initial screening in patients of the outpatient clinic of the Department of Transfusion Medicine and Hemostaseology of the University Hospital Erlangen, Germany, revealed 183 patients with a history of CVT. An initial screening identified a number of common prothrombic risk factors, including Factor V Leiden (rs6025) and Prothrombin G20210A (rs1799963). All patients without relevant findings (58 individuals) were invited to participate in a subsequent genetic analysis of 55 relevant genes using next-generation sequencing (NGS). Three intron variants (ADAMTS13: rs28446901, FN1: rs56380797, rs35343655) were identified to occur with a significantly higher frequency in the CVT patient cohort compared to the general European population. Furthermore, the combined prevalence of at least two of four potentially prothrombic variants (FGA (rs6050), F13A1 (rs5985), ITGB3 (rs5918), and PROCR (rs867186)) was significantly higher in the CVT subjects. The possible impact of the identified variants on CVT is discussed.

Sympathetic Input to Multiple Cell Types in Mouse and Human Colon Produces Region-Specific Responses.

BACKGROUND & AIMS: The colon is innervated by intrinsic and extrinsic neurons that coordinate functions necessary for digestive health. Sympathetic input suppresses colon motility by acting on intrinsic myenteric neurons, but the extent of sympathetic-induced changes on large-scale network activity in myenteric circuits has not been determined. Compounding the complexity of sympathetic function, there is evidence that sympathetic transmitters can regulate activity in non-neuronal cells (such as enteric glia and innate immune cells). METHODS: We performed anatomical tracing, immunohistochemistry, optogenetic (GCaMP calcium imaging, channelrhodopsin), and colon motility studies in mice and single-cell RNA sequencing in human colon to investigate how sympathetic postganglionic neurons modulate colon function. RESULTS: Individual neurons in each sympathetic prevertebral ganglion innervated the proximal or distal colon, with processes closely opposed to multiple cell types. Calcium imaging in semi-intact mouse colon preparations revealed changes in spontaneous and evoked neural activity, as well as activation of non-neuronal cells, induced by sympathetic nerve stimulation. The overall pattern of response to sympathetic stimulation was unique to the proximal or distal colon. Region-specific changes in cellular activity correlated with motility patterns produced by electrical and optogenetic stimulation of sympathetic pathways. Pharmacology experiments (mouse) and RNA sequencing (human) indicated that appropriate receptors were expressed on different cell types to account for the responses to sympathetic stimulation. Regional differences in expression of α-1 adrenoceptors in human colon emphasize the translational relevance of our mouse findings. CONCLUSIONS: Sympathetic neurons differentially regulate activity of neurons and non-neuronal cells in proximal and distal colon to promote distinct changes in motility patterns, likely reflecting the distinct roles played by these 2 regions.

Spectroscopic and in†vitro Investigations of Fe2+ /α-Ketoglutarate-Dependent Enzymes Involved in Nucleic Acid Repair and Modification.

The activation of molecular oxygen for the highly selective functionalization and repair of DNA and RNA nucleobases is achieved by α-ketoglutarate (α-KG)/iron-dependent dioxygenases. Of special interest are the human homologues AlkBH of Escherichia coli EcAlkB and ten-eleven translocation (TET) enzymes. These enzymes are involved in demethylation or dealkylation of DNA and RNA, although additional physiological functions are continuously being found. Given their importance, studying enzyme-substrate interactions, turnover and kinetic parameters is pivotal for the understanding of the mode of action of these enzymes. Diverse analytical methods, including X-ray crystallography, UV/Vis absorption, electron paramagnetic resonance (EPR), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy have been employed to study the changes in the active site and the overall enzyme structure upon substrate, cofactor, and inhibitor addition. Several methods are now available to assess the activity of these enzymes. By discussing limitations and possibilities of these techniques for EcAlkB, AlkBH and TET we aim to give a comprehensive synopsis from a bioinorganic point-of-view, addressing researchers from different disciplines working in the highly interdisciplinary and rapidly evolving field of epigenetic processes and DNA/RNA repair and modification.

Carrier Protein-Free Enzymatic Biaryl Coupling in Arylomycin A2 Assembly and Structure of the Cytochrome P450 AryC.

Invited for the cover of this issue are Sabine Schneider, Tobias A. M. Gulder and co-workers at Technical University of Dresden, Technical University of Munich and Ludwig-Maximillians-University Munich. The image depicts the crystal structure of the cytochrome P450 AryC from arylomycin biosynthesis. Read the full text of the article at 10.1002/chem.202103389.

Carrier Protein-Free Enzymatic Biaryl Coupling in Arylomycin A2 Assembly and Structure of the Cytochrome P450 AryC.

The arylomycin antibiotics are potent inhibitors of bacterial type I signal peptidase. These lipohexapeptides contain a biaryl structural motif reminiscent of glycopeptide antibiotics. We herein describe the functional and structural evaluation of AryC, the cytochrome P450 performing biaryl coupling in biosynthetic arylomycin assembly. Unlike its enzymatic counterparts in glycopeptide biosynthesis, AryC converts free substrates without the requirement of any protein interaction partner, likely enabled by a strongly hydrophobic cavity at the surface of AryC pointing to the substrate tunnel. This activity enables chemo-enzymatic assembly of arylomycin A2 that combines the advantages of liquid- and solid-phase peptide synthesis with late-stage enzymatic cross-coupling. The reactivity of AryC is unprecedented in cytochrome P450-mediated biaryl construction in non-ribosomal peptides, in which peptidyl carrier protein (PCP)-tethering so far was shown crucial both in vivo and in vitro.

Neurodevelopmental toxicity assessment of flame retardants using a human DNT in vitro testing battery.

Due to their neurodevelopmental toxicity, flame retardants (FRs) like polybrominated diphenyl ethers are banned from the market and replaced by alternative FRs, like organophosphorus FRs, that have mostly unknown toxicological profiles. To study their neurodevelopmental toxicity, we evaluated the hazard of several FRs including phased-out polybrominated FRs and organophosphorus FRs: 2,2',4,4'-tetrabromodiphenylether (BDE-47), 2,2',4,4',5-pentabromodiphenylether (BDE-99), tetrabromobisphenol A, triphenyl phosphate, tris(2-butoxyethyl) phosphate and its metabolite bis-(2-butoxyethyl) phosphate, isodecyl diphenyl phosphate, triphenyl isopropylated phosphate, tricresyl phosphate, tris(1,3-dichloro-2-propyl) phosphate, tert-butylphenyl diphenyl phosphate, 2-ethylhexyl diphenyl phosphate, tris(1-chloroisopropyl) phosphate, and tris(2-chloroethyl) phosphate. Therefore, we used a human cell-based developmental neurotoxicity (DNT) in vitro battery covering a large variety of neurodevelopmental endpoints. Potency according to the respective most sensitive benchmark concentration (BMC) across the battery ranked from <1 µM (5 FRs), 1<10 µM (7 FRs) to the >10 µM range (3 FRs). Evaluation of the data with the ToxPi tool revealed a distinct ranking (a) than with the BMC and (b) compared to the ToxCast data, suggesting that DNT hazard of these FRs is not well predicted by ToxCast assays. Extrapolating the DNT in vitro battery BMCs to human FR exposure via breast milk suggests low risk for individual compounds. However, it raises a potential concern for real-life mixture exposure, especially when different compounds converge through diverse modes-of-action on common endpoints, like oligodendrocyte differentiation in this study. This case study using FRs suggests that human cell-based DNT in vitro battery is a promising approach for neurodevelopmental hazard assessment and compound prioritization in risk assessment.

Suppression of SARS-CoV-2 Replication with Stabilized and Click-Chemistry Modified siRNAs.

The emergence of more transmissible or aggressive variants of SARS-CoV-2 requires the development of antiviral medication that is quickly adjustable to evolving viral escape mutations. Here we report the synthesis of chemically stabilized small interfering RNA (siRNA) against SARS-CoV-2. The siRNA can be further modified with receptor ligands such as peptides using CuI -catalysed click-chemistry. We demonstrate that optimized siRNAs can reduce viral loads and virus-induced cytotoxicity by up to five orders of magnitude in cell lines challenged with SARS-CoV-2. Furthermore, we show that an ACE2-binding peptide-conjugated siRNA is able to reduce virus replication and virus-induced apoptosis in 3D mucociliary lung microtissues. The adjustment of the siRNA sequence allows a rapid adaptation of their antiviral activity against different variants of concern. The ability to conjugate the siRNA via click-chemistry to receptor ligands facilitates the construction of targeted siRNAs for a flexible antiviral defence strategy.

Wnt5a is a transcriptional target of Gli3 and Trps1 at the onset of chondrocyte hypertrophy.

During endochondral ossification, the differentiation of proliferating into hypertrophic chondrocytes is a key step determining the pace of bone formation and the future length of the skeletal elements. A variety of transcription factors are expressed at the onset of hypertrophy coordinating the expression of different signaling molecules like Bmps, Ihh and Wnt proteins. In this study, we characterized the murine Wnt5a promoter and provide evidence that two alternative Wnt5a transcripts, Ts1 and Ts2, are differentially expressed in the developing skeletal elements. Ts2 expression decreases while Ts1 expression increases during chondrocyte differentiation. The transcription factor Trps1 and the activator form of Gli3 (Gli3A), which is a mediator of Hedgehog signaling, activate Wnt5a expression. In Chromatin Immunoprecipitation and reporter gene assays, we identified two upstream regulatory sequences (URS) in the Wnt5a promoter mediating either activating or repressive functions. The activating URS1 is bound by Trps1 and Gli3A in vitro and in vivo to upregulate Wnt5a expression. Loss of both transcription factors decreases endogenous Wnt5a mRNA and protein levels during chondrocyte differentiation, thereby identifying Wnt5a as a target gene of Trps1 and Gli3A in chondrocytes.

Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and C. elegans.

The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N3-methylcytosine (m3C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N1-methyladenosine (m1A) in tRNA or formation of 5-formyl cytosine (f5C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPRmt) in C. elegans.

Glial Cell-Derived Neurotrophic Factor Induces Enteric Neurogenesis and Improves Colon Structure and Function in Mouse Models of Hirschsprung Disease.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is a life-threatening birth defect in which the distal colon is devoid of enteric neural ganglia. HSCR is treated by surgical removal of aganglionic bowel, but many children continue to have severe problems after surgery. We studied whether administration of glial cell derived neurotrophic factor (GDNF) induces enteric nervous system regeneration in mouse models of HSCR. METHODS: We performed studies with four mouse models of HSCR: Holstein (HolTg/Tg, a model for trisomy 21-associated HSCR), TashT (TashTTg/Tg, a model for male-biased HSCR), Piebald-lethal (Ednrbs-l//s-l, a model for EDNRB mutation-associated HSCR), and Ret9/- (with aganglionosis induced by mycophenolate). Mice were given rectal enemas containing GDNF or saline (control) from postnatal days 4 through 8. We measured survival times of mice, and colon tissues were analyzed by histology, immunofluorescence, and immunoblots. Neural ganglia regeneration and structure, bowel motility, epithelial permeability, muscle thickness, and neutrophil infiltration were studied in colon tissues and in mice. Stool samples were collected, and microbiomes were analyzed by 16S rRNA gene sequencing. Time-lapse imaging and genetic cell-lineage tracing were used to identify a source of GDNF-targeted neural progenitors. Human aganglionic colon explants from children with HSCR were cultured with GDNF and evaluated for neurogenesis. RESULTS: GDNF significantly prolonged mean survival times of HolTg/Tg mice, Ednrbs-l//s-l mice, and male TashTTg/Tg mice, compared with control mice, but not Ret9/- mice (which had mycophenolate toxicity). Mice given GDNF developed neurons and glia in distal bowel tissues that were aganglionic in control mice, had a significant increase in colon motility, and had significant decreases in epithelial permeability, muscle thickness, and neutrophil density. We observed dysbiosis in fecal samples from HolTg/Tg mice compared with feces from wild-type mice; fecal microbiomes of mice given GDNF were similar to those of wild-type mice except for Bacteroides. Exogenous luminal GDNF penetrated aganglionic colon epithelium of HolTg/Tg mice, inducing production of endogenous GDNF, and new enteric neurons and glia appeared to arise from Schwann cells within extrinsic nerves. GDNF application to cultured explants of human aganglionic bowel induced proliferation of Schwann cells and formation of new neurons. CONCLUSIONS: GDNF prolonged survival, induced enteric neurogenesis, and improved colon structure and function in 3 mouse models of HSCR. Application of GDNF to cultured explants of aganglionic bowel from children with HSCR induced proliferation of Schwann cells and formation of new neurons. GDNF might be developed for treatment of HSCR.

Robust, 3-Dimensional Visualization of Human Colon Enteric Nervous System Without Tissue Sectioning.

BACKGROUND & AIMS: Small, 2-dimensional sections routinely used for human pathology analysis provide limited information about bowel innervation. We developed a technique to image human enteric nervous system (ENS) and other intramural cells in 3 dimensions. METHODS: Using mouse and human colon tissues, we developed a method that combines tissue clearing, immunohistochemistry, confocal microscopy, and quantitative analysis of full-thickness bowel without sectioning to quantify ENS and other intramural cells in 3 dimensions. RESULTS: We provided 280 adult human colon confocal Z-stacks from persons without known bowel motility disorders. Most of our images were of myenteric ganglia, captured using a 20× objective lens. Full-thickness colon images, viewed with a 10× objective lens, were as large as 4 × 5 mm2. Colon from 2 pediatric patients with Hirschsprung disease was used to show distal colon without enteric ganglia, as well as a transition zone and proximal pull-through resection margin where ENS was present. After testing a panel of antibodies with our method, we identified 16 antibodies that bind to molecules in neurons, glia, interstitial cells of Cajal, and muscularis macrophages. Quantitative analyses demonstrated myenteric plexus in 24.5% ± 2.4% of flattened colon Z-stack area. Myenteric ganglia occupied 34% ± 4% of myenteric plexus. Single myenteric ganglion volume averaged 3,527,678 ± 573,832 mm3 with 38,706 ± 5763 neuron/mm3 and 129,321 ± 25,356 glia/mm3. Images of large areas provided insight into why published values of ENS density vary up to 150-fold-ENS density varies greatly, across millimeters, so analyses of small numbers of thin sections from the same bowel region can produce varying results. Neuron subtype analysis revealed that approximately 56% of myenteric neurons stained with neuronal nitric oxide synthase antibody and approximately 33% of neurons produce and store acetylcholine. Transition zone regions from colon tissues of patients with Hirschsprung disease had ganglia in multiple layers and thick nerve fiber bundles without neurons. Submucosal neuron distribution varied among imaged colon regions. CONCLUSIONS: We developed a 3-dimensional imaging method for colon that provides more information about ENS structure than tissue sectioning. This approach could improve diagnosis for human bowel motility disorders and may be useful for other bowel diseases as well.

Oral or Topical Pain Therapy-How Would Patients Decide? A Discrete Choice Experiment in Patients with Peripheral Neuropathic Pain.

To ensure an adequate pain therapy with high patient adherence, it is necessary to know and consider patient preferences. A discrete choice experiment was used to obtain patients' preferences regarding treatment with systemic or topical pain medication. Patients with peripheral neuropathic pain (pNP) were recruited in two pain-focused practices in Germany. To identify relevant attributes of topical or systemic pain medication, a literature review and face-to-face interviews with experts for pain treatment were conducted. The attributes used in the choice scenarios were noticeable onset of effect, time spent in medical office, risk of systemic and local side effects, and impairment of daily life with regard to sleep quality and sexuality. The model was estimated with a mixed multinomial logit regression model. The study included 153 participants suffering from moderate to severe pNP. Most important attributes from patient's perspective was noticeable onset of effect (odds ratio 2.141 [95% confidence interval 1.837 to 2.494]), followed by risk of systemic side effects (2.038 [1.731 to 2.400]) and risk of sexual dysfunction (1.839 [1.580 to 2.140]), while risk of local side effects regarding skin ranked fourth (1.612 [1.321 to 1.966]). The impairment of sleep quality was also significant but less important (1.556 [1.346 to 1.798]). Local side effects were more likely to be accepted than systemic side effects. The risk of sexual dysfunction as a side effect of treatment is very important for patients, although it has received little attention in the literature.