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Author(s): A. Sentrk [1]; S. Pfennig [1]; A. Weiss [1]; K. Burk [1]; A. Acker-Palmer (corresponding author) [1] replying to T. Pohlkamp et al . Nature 539, http://dx.doi.org/10.1038/nature20129 (2016). In [...]

Background and Aims: We analyzed the prevalence of concomitant intestinal stenosis in patients with fistulizing Crohn's disease (CD), including the NOD2/CARD15 and IL23R genotype status.Methods: Medical records of n = 1,110 patients with inflammatory bowel diseases were screened for patients with fistulizing and stricturing CD. Study inclusion required diagnosis of stenosis made within 6 months of diagnosing fistulas. CD-associated NOD2 and IL23R variants were genotyped. Similarly, we prospectively investigated 42 patients presenting with fistulizing CD.Results: In the retrospective study (n = 333 CD patients), fistulas were found in 145 (43.5%) patients and stenoses in 223 (67.0%) patients. Concomitant stenosis was diagnosed in 125 patients with fistulas resulting in a positive predictive value (PPV) of 86.2% for fistulas predicting intestinal stenosis (p = 5.53 x 10(-11); OR 5.74, 95% CI 3.22-10.50). In logistic regression analysis, presence of fistulas (OR 4.51; 95% CI 2.54-8.01, p = 2.68 x 10(-7)) and disease duration (OR 1.09; 95% CI 1.05-1.13; p = 3.19 x 10(-6)) were strongly associated with intestinal stenosis. NOD2 genotype information, but not IL23R status, increased the PPV for the correct diagnosis of stenosis (PPV = 89.9%). All homozygous carriers (100%) of NOD2 variants with fistulizing CD were diagnosed with stenosis; 1007fs homozygotes were found more often among patients with fistulas and stenoses than in patients without stenoses and fistulas (p = 0.00037). Similar results were found in the prospective analysis, in which 83.3% of the patients with fistulizing CD had concomitant stenosis.Conclusion: Fistulizing CD is strongly associated with concomitant intestinal stenosis, particularly in homozygous carriers of NOD2 mutations. [ABSTRACT FROM AUTHOR]

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We found that PRC1 is essential for the specification of segmentally restricted spinal motor neuron (MN) subtypes, while PRC2 activity is dispensable to maintain MN positional identities during terminal differentiation. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox-dependent specification programs by derepressed Hox13 paralogs ( Hoxa13 , Hoxb13 , Hoxc13 , Hoxd13 ). These results indicate that PRC1 can function in the absence of de novo PRC2-dependent histone methylation to maintain chromatin topology and postmitotic neuronal fate., Competing Interests: AS, SP, MB, AM, AK, EM, JD No competing interests declared, (© 2022, Sawai et al.)

The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

ATP-dependent nucleosome remodeling factors sculpt the nucleosomal landscape of eukaryotic chromatin. They deposit or evict nucleosomes or reposition them along DNA in a process termed nucleosome sliding. Remodeling has traditionally been analyzed using mononucleosomes as a model substrate. In vivo, however, nucleosomes form extended arrays with regular spacing. Here, we describe how regularly spaced nucleosome arrays can be reconstituted in vitro and how these arrays can be used to dissect remodeling in the test tube. We outline two assays. The first assay senses various structural changes to a specific nucleosome within the nucleosomal array whereas the second assay is specific toward detecting repositioning of nucleosomes within the array. Both assays exploit changes to the accessibility of DNA to restriction enzymes during the remodeling reaction.

Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in response to neuronal activity is critical for synaptic function and plasticity. Here, we show that neuronal activity induces the binding of ephrinB2 and ApoER2 receptors at the postsynapse to regulate de novo insertion of AMPA receptors. Mechanistically, the multi-PDZ adaptor glutamate-receptor-interacting protein 1 (GRIP1) binds ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors. Phosphorylation of ephrinB2 in a serine residue (Ser-9) is essential for the stability of such a complex. In vivo, a mutation on ephrinB2 Ser-9 in mice results in a complete disruption of the complex, absence of ApoER2 downstream signaling, and impaired activity-induced and ApoER2-mediated AMPA receptor insertion. Using compound genetics, we show the requirement of this complex for long-term potentiation (LTP). Together, our findings uncover a cooperative ephrinB2 and ApoER2 signaling at the synapse, which serves to modulate activity-dependent AMPA receptor dynamic changes during synaptic plasticity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

ISWI-family nucleosome remodeling enzymes need the histone H4 N-terminal tail to mobilize nucleosomes. Here we mapped the H4-tail binding pocket of ISWI. Surprisingly the binding site was adjacent to but not overlapping with the docking site of an auto-regulatory motif, AutoN, in the N-terminal region (NTR) of ISWI, indicating that AutoN does not act as a simple pseudosubstrate as suggested previously. Rather, AutoN cooperated with a hitherto uncharacterized motif, termed AcidicN, to confer H4-tail sensitivity and discriminate between DNA and nucleosomes. A third motif in the NTR, ppHSA, was functionally required in vivo and provided structural stability by clamping the NTR to Lobe 2 of the ATPase domain. This configuration is reminiscent of Chd1 even though Chd1 contains an unrelated NTR. Our results shed light on the intricate structural and functional regulation of ISWI by the NTR and uncover surprising parallels with Chd1., Competing Interests: The authors declare that no competing interests exist.

D-Alanylation of lipoteichoic acids plays an important role in modulating the properties of Gram-positive bacteria cell walls. The D-alanyl carrier protein DltC from Bacillus subtilis has been solved in apo- and two cofactor-modified holo-forms, whereby the entire phosphopantetheine moiety is defined in one. The atomic resolution of the apo-structure allows delineation of alternative conformations within the hydrophobic core of the 78 residue four helix bundle. In contrast to previous reports for a peptidyl carrier protein from a non-ribosomal peptide synthetase, no obvious structural differences between apo- and holo-DltC forms are observed. Solution NMR spectroscopy confirms these findings and demonstrates in addition that the two forms exhibit similar backbone dynamics on the ps-ns and ms timescales., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.

In Costa Rica, thousands of tones of agricultural pesticides have been used for decades and their use is continuously increasing due to intensive and expanding production of coffee, pineapple, rice, ornamental plants and bananas. The objective of this study was to evaluate whether choline esterase (ChE) activity could be used as a biomarker of exposure to pesticides in the Costa Rican native fish Astyanax aeneus (characidae). Three methods used in order to evaluate the ChE biomarker were as follows: Laboratory studies where A. aeneus was exposed to organophosphate pesticide (ethoprophos); In situ 48 hr exposure assessment using caging experiments with fish exposed upstream and downstream of banana plantations and ChE activity estimation of in fish captured directly at sites with different degrees of pesticide exposure. Results from the laboratory studies showed that ChE activity in both brain and muscle tissue was significantly lower in fish exposed to ethoprophos than in controls. Fish from the caging experiments showed no difference in ChE activity neither in brain nor in muscle tissue between the four tested sites and was attributed to the short duration of the exposure. Asignificant difference in ChE activity was determined in muscle of fish captured from Laguna Madre de Dios compared to fish from Canal Batán. Although our laboratory results revealed that ChE activity in A. aeneus was highly responsive to ethoprophos, results from field experiments were less conclusive and showed that the captured fish showed large variability in ChE activity and that more research is needed before ChE activity can be used as reliable biomarker of pesticide exposure.

Current knowledge on fate and effect of agricultural pesticides comes is mainly from temperate ecosystems. More studies are needed in tropical systems in order to assess contamination risks to nontarget endemic tropical species from the extensive use of pesticides e.g. in banana and pineapple plantations. In this study, acute laboratory toxicity tests with organophosphate pesticides ethoprophos and chlorpyrifos were conducted on two Costa Rican species, cladoceran Daphnia ambigua and fish Parachromis dovii. Tests showed that chlorpyrifos was more toxic than ethoprophos to D. ambigua and P. dovii and that D. ambigua was also more sensitive than P. dovii to both pesticides. Additionally, bioassays were performed by exposing D. magna and P. dovii to contaminated water collected from the field. Chemical analyses of field water revealed that fungicides were generally the most frequent pesticide group found, followed by insecticides/nematicides and herbicides. The bioassays and values obtained from the literature confirmed that D. magna was more sensitive to pesticide contamination than P. dovii and that D. ambigua was more sensitive than D. magna, suggesting that the native cladoceran is a more suitable test species than its temperate counterpart. Species sensitivity distributions showed no significant difference in sensitivity between tropical and temperate fish and the arthropod species exposed to chlorpyrifos in this study. Choline esterase activity (ChE) was measured in P. dovii in laboratory tests in order to assess the applicability of this biomarker. ChE inhibition in P. dovii was observed in the laboratory at levels below the LC10 of both ethoprophos and chlorpyrifos, confirming that ChE is an efficient biomarker of exposure. Both indigenous Costa Rican species used in this study were found to be suitable standard tropical test species. Further studies are needed to investigate how protective the safe environmental concentrations, derived from LC50 of native tropical species, are for protecting tropical aquatic natural communities.

Calmodulin (CaM) is a highly conserved intracellular Ca(2+)-binding protein that exerts important functions in many cellular processes. Prominent examples of CaM-regulated proteins are adenylyl cyclases (ACs), which synthesize cAMP as a central second messenger. The interaction of ACs with CaM represents the link between Ca(2+)-signaling and cAMP-signaling pathways. Thereby, different AC isoforms stimulated by CaM, comprise diverse mechanisms of regulation by the Ca(2+) sensor. To extend the structural information about the detailed mechanisms underlying the regulation of AC8 by CaM, we employed an integrated approach combining chemical cross-linking and mass spectrometry with two peptides representing the CaM-binding regions of AC8. These experiments reveal that the structures of CaM/AC8 peptide complexes are similar to that of the CaM/skeletal muscle myosin light chain kinase peptide complex where CaM is collapsed around the target peptide that binds to CaM in an antiparallel orientation. Cross-linking experiments were complemented by investigating the binding of AC8 peptides to CaM thermodynamically with isothermal titration calorimetry. There were no hints on a complex, in which both AC8 peptides bind simultaneously to CaM, refining our current understanding of the interaction between CaM and AC8.

Biomarkers are a widely applied approach in environmental studies. Analyses of cholinesterase (ChE), glutathione S-transferase (GST) and lipid peroxidation (LPO) are biomarkers that can provide information regarding early effects of pollutants at different biochemical levels on an organism. The aim of this study was to evaluate the biomarker approach on a Costa Rican native and relevant species. For this, larvae of gar (Atractosteus tropicus) were exposed to the organophosphorus nematicide, ethoprophos. Acute (96hr) exposure was conducted with pesticide concentrations ranging from 0.1 microg/L to 1 500 microg/L. The 96hr LC50 calculated was 859.7 microg/L. After exposure, three biomarkers (ChE, GST and LPO) were analyzed in fish that survived the acute test. The lowest observed effect concentration (LOEC) regarding ChE activity inhibition was 50 microg/L. This concentration produced a significant inhibition (p<0.05) of the enzyme by 20%. The highest concentration tested without showing any effect on ChE activity and therefore considered as no observed effect concentration (NOEC) was 10 microg/L. Ethoprophos concentration of 400 microg/L caused a ChE inhibition by 79%. In this study, no significant variations (p>0.05) in GST activity and LPO were observed in A. tropicus larvae after exposure to ethoprophos.

Re-engineering of nonribosomal peptide synthetase molecular assembly lines has been hampered by a lack of detailed knowledge concerning inter-domain substrate transfer. Recent structural studies of catalytically relevant domain-domain interactions provide valuable insights into this problem (Liu et al., 2011; Sundlov et al., 2012 [in this issue of Chemistry & Biology])., (Copyright © 2012 Elsevier Ltd. All rights reserved.)