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Infection by is the primary cause of gastric adenocarcinoma. The most potent virulence factor is cytotoxin-associated gene A (CagA), which is translocated by a type 4 secretion system (T4SS) into gastric epithelial cells and activates oncogenic signaling pathways. The gene encodes for a key component of the T4SS and can undergo gene rearrangements. We have shown that the cancer chemopreventive agent α-difluoromethylornithine (DFMO), known to inhibit the enzyme ornithine decarboxylase, reduces -mediated gastric cancer incidence in Mongolian gerbils. In the present study, we questioned whether DFMO might directly affect pathogenicity. We show that output strains isolated from gerbils treated with DFMO exhibit reduced ability to translocate CagA in gastric epithelial cells. Further, we frequently detected genomic modifications in the middle repeat region of the gene of output strains from DFMO-treated animals, which were associated with alterations in the CagY protein. Gerbils did not develop carcinoma when infected with a DFMO output strain containing rearranged or the parental strain in which the wild-type was replaced by with DFMO-induced rearrangements. Lastly, we demonstrate that in vitro treatment of by DFMO induces oxidative DNA damage, expression of the DNA repair enzyme MutS2, and mutations in , demonstrating that DFMO directly affects genomic stability. Deletion of abrogated the ability of DFMO to induce rearrangements directly. In conclusion, DFMO-induced oxidative stress in leads to genomic alterations and attenuates virulence.
Annexin proteins function as Ca-dependent regulators of membrane trafficking and repair that may also modulate membrane curvature. Here, using high-resolution confocal imaging, we report that the intestine-specific annexin A13 (ANX A13) localizes to the tips of intestinal microvilli and determined the crystal structure of the ANX A13a isoform to 2.6 Å resolution. The structure revealed that the N terminus exhibits an alternative fold that converts the first two helices and the associated helix-loop-helix motif into a continuous α-helix, as stabilized by a domain-swapped dimer. We also found that the dimer is present in solution and partially occludes the membrane-binding surfaces of annexin, suggesting that dimerization may function as a means for regulating membrane binding. Accordingly, as revealed by binding and cellular localization assays, ANX A13a variants that favor a monomeric state exhibited increased membrane association relative to variants that favor the dimeric form. Together, our findings support a mechanism for how the association of the ANX A13a isoform with the membrane is regulated.
© 2019 McCulloch et al.
The regulatory subunit of human DNA primase has a C-terminal domain (p58C) that contains a [4Fe4S] cluster and binds DNA. Previous electrochemical analysis of a p58C construct revealed that its affinity for DNA is sensitive to the redox state of the [4Fe4S] cluster. Concerns about the validity of this conclusion have been raised, based in part on differences in X-ray crystal structures of the p58C272-464 construct used for that study and that of a N-terminally shifted p58C266-456 construct and consequently, an assumption that p58C272-464 has abnormal physical and functional properties. To address this controversy, a new p58C266-464 construct containing all residues was crystallized under the conditions previously used for crystallizing p58C272-464, and the solution structures of both constructs were assessed using circular dichroism and NMR spectroscopy. In the new crystal structure, p58C266-464 exhibits the same elements of secondary structure near the DNA binding site as observed in the crystal structure of p58C272-464. Moreover, in solution, circular dichroism and 15N,1H-heteronuclear single quantum coherence (HSQC) NMR spectra show there are no significant differences in the distribution of secondary structures or in the tertiary structure or the two constructs. To validate that the two constructs have the same functional properties, binding of a primed DNA template was measured using a fluorescence-based DNA binding assay, and the affinities for this substrate were the same (3.4 ± 0.5 μM and 2.7 ± 0.3 μM, respectively). The electrochemical properties of p58C266-464 were also measured and this p58C construct was able to engage in redox switching on DNA with the same efficiency as p58C272-464. Together, these results show that although p58C can be stabilized in different conformations in the crystalline state, in solution there is effectively no difference in the structure and functional properties of p58C constructs of different lengths.
The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.
© 2018, Fenix et al.
CagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation among strains in the steady-state levels of CagA and that a strain-specific motif downstream of the transcriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. The 5' untranslated region contains a predicted stem-loop-forming structure adjacent to the +59 motif. In the current study, we investigated the effect of the +59 motif and the adjacent stem-loop on transcript levels and mRNA stability. Using site-directed mutagenesis, we found that mutations predicted to disrupt the stem-loop structure resulted in decreased steady-state levels of both the transcript and the CagA protein. Additionally, these mutations resulted in a decreased mRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-state transcript and CagA protein levels but did not affect transcript stability. transcript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augment transcript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in the 5' untranslated region influence the levels of expression.
Copyright © 2019 American Society for Microbiology.
Diffusion MRI (dMRI) fiber tractography has become a pillar of the neuroimaging community due to its ability to noninvasively map the structural connectivity of the brain. Despite widespread use in clinical and research domains, these methods suffer from several potential drawbacks or limitations. Thus, validating the accuracy and reproducibility of techniques is critical for sound scientific conclusions and effective clinical outcomes. Towards this end, a number of international benchmark competitions, or "challenges", has been organized by the diffusion MRI community in order to investigate the reliability of the tractography process by providing a platform to compare algorithms and results in a fair manner, and evaluate common and emerging algorithms in an effort to advance the state of the field. In this paper, we summarize the lessons from a decade of challenges in tractography, and give perspective on the past, present, and future "challenges" that the field of diffusion tractography faces.
Copyright © 2018 Elsevier Inc. All rights reserved.
The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate this environment, we generated a nine-strain actinomycete community and used 16S rDNA sequencing to deconvolute the stochastic production of antimicrobial activity that was not observed from any of the axenic cultures. We subsequently simplified the community to just two strains and identified sp. AA4 as the producing strain and M145 as an inducing strain. Bioassay-guided isolation identified amycomicin (AMY), a highly modified fatty acid containing an epoxide isonitrile warhead as a potent and specific inhibitor of Amycomicin targets an essential enzyme (FabH) in fatty acid biosynthesis and reduces infection in a mouse skin-infection model. The discovery of AMY demonstrates the utility of screening complex communities against specific targets to discover small-molecule antibiotics.
Endothelial dysfunction is a known consequence of bone morphogenetic protein type II receptor () mutations seen in pulmonary arterial hypertension (PAH). However, standard 2D cell culture models fail to mimic the mechanical environment seen in the pulmonary vasculature. Hydrogels have emerged as promising platforms for 3D disease modeling due to their tunable physical and biochemical properties. In order to recreate the mechanical stimuli seen in the pulmonary vasculature, we have created a novel 3D hydrogel-based pulmonary vasculature model ("artificial arteriole") that reproduces the pulsatile flow rates and pressures seen in the human lung. Using this platform, we studied both and WT endothelial cells to better understand how the addition of oscillatory flow and physiological pressure influenced gene expression, cell morphology, and cell permeability. The addition of oscillatory flow and pressure resulted in several gene expression changes in both WT and cells. However, for many pathways with relevance to PAH etiology, cells responded differently when compared to the WT cells. cells were also found not to elongate in the direction of flow, and instead remained stagnant in morphology despite mechanical stimuli. The increased permeability of the layer was successfully reproduced in our artificial arteriole, with the addition of flow and pressure not leading to significant changes in permeability. Our artificial arteriole is the first to model many mechanical properties seen in the lung. Its tunability enables several new opportunities to study the endothelium in pulmonary vascular disease with increased control over environmental parameters.
The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.
DA closure is crucial for the transition from fetal to neonatal life. This closure is supported by changes to the DA's signaling and structural properties that distinguish it from neighboring vessels. Examining transcriptional differences between these vessels is key to identifying genes or pathways responsible for DA closure. Several microarray studies have explored the DA transcriptome in animal models but varied experimental designs have led to conflicting results. Thorough transcriptomic analysis of the human DA has yet to be performed. A clear picture of the DA transcriptome is key to guiding future research endeavors, both to allow more targeted treatments in the clinical setting, and to understand the basic biology of DA function. In this review, we use a cross-species cross-platform analysis to consider all available published rodent microarray data and novel human RNAseq data in order to provide high priority candidate genes for consideration in future DA studies.
Copyright © 2018 Elsevier Inc. All rights reserved.