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Gene network transitions in embryos and other fate-changing contexts involve combinations of transcription factors. A subset of fate-changing transcription factors act as pioneers; they scan and target nucleosomal DNA and initiate cooperative events that can open the local chromatin. However, a gap has remained in understanding how molecular interactions with the nucleosome contribute to the chromatin-opening phenomenon. Here we identified a short α-helical region, conserved among FOXA pioneer factors, that interacts with core histones and contributes to chromatin opening in vitro. The same domain is involved in chromatin opening in early mouse embryos for normal development. Thus, local opening of chromatin by interactions between pioneer factors and core histones promotes genetic programming.
Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith, 8, e73204 (2013); S. Kozar , 13, 626-633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogen we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.
Background - Helper T cell activity is dysregulated in a number of diseases including those associated with rheumatic autoimmunity. Treatment options are limited and usually consist of systemic immune suppression, resulting in undesirable consequences from compromised immunity. Hedgehog (Hh) signaling has been implicated in the activation of T cells and the formation of the immune synapse, but remains understudied in the context of autoimmunity. Modulation of Hh signaling has the potential to enable controlled immunosuppression but a potential therapy has not yet been developed to leverage this opportunity.
Methods - In this work, we developed biodegradable nanoparticles to enable targeted delivery of eggmanone (Egm), a specific Hh inhibitor, to CD4 T cell subsets. We utilized two FDA-approved polymers, poly(lactic-co-glycolic acid) and polyethylene glycol, to generate hydrolytically degradable nanoparticles. Furthermore, we employed maleimide-thiol mediated conjugation chemistry to decorate nanoparticles with anti-CD4 F(ab') antibody fragments to enable targeted delivery of Egm.
Results - Our novel delivery system achieved a highly specific association with the majority of CD4 T cells present among a complex cell population. Additionally, we have demonstrated antigen-specific inhibition of CD4 T cell responses mediated by nanoparticle-formulated Egm.
Conclusion - This work is the first characterization of Egm's immunomodulatory potential. Importantly, this study also suggests the potential benefit of a biodegradable delivery vehicle that is rationally designed for preferential interaction with a specific immune cell subtype for targeted modulation of Hh signaling.
© 2020 Haycook et al.
Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.
Copyright © 2020 Elsevier Inc. All rights reserved.
Increased endothelial permeability is central to the pathogenesis of sepsis and leads to organ dysfunction and death but the endogenous mechanisms that drive increased endothelial permeability are not completely understood. We previously reported that cell-free hemoglobin (CFH), elevated in 80% of patients with sepsis, increases lung microvascular permeability in an ex vivo human lung model and cultured endothelial cells. In this study, we augmented a murine model of polymicrobial sepsis with elevated circulating CFH to test the hypothesis that CFH increases microvascular endothelial permeability by inducing endothelial apoptosis. Mice were treated with an intraperitoneal injection of cecal slurry with or without a single intravenous injection of CFH. Severity of illness, mortality, systemic and lung inflammation, endothelial injury and dysfunction and lung apoptosis were measured at selected time points. We found that CFH added to CS increased sepsis mortality, plasma inflammatory cytokines as well as lung apoptosis, edema and inflammation without affecting large vessel reactivity or vascular injury marker concentrations. These results suggest that CFH is an endogenous mediator of increased endothelial permeability and apoptosis in sepsis and may be a promising therapeutic target.
Functional coupling between the amygdala and the dorsomedial prefrontal cortex (dmPFC) has been implicated in the generation of negative affective states; however, the mechanisms by which stress increases amygdala-dmPFC synaptic strength and generates anxiety-like behaviors are not well understood. Here, we show that the mouse basolateral amygdala (BLA)-prelimbic prefrontal cortex (plPFC) circuit is engaged by stress and activation of this pathway in anxiogenic. Furthermore, we demonstrate that acute stress exposure leads to a lasting increase in synaptic strength within a reciprocal BLA-plPFC-BLA subcircuit. Importantly, we identify 2-arachidonoylglycerol (2-AG)-mediated endocannabinoid signaling as a key mechanism limiting glutamate release at BLA-plPFC synapses and the functional collapse of multimodal 2-AG signaling as a molecular mechanism leading to persistent circuit-specific synaptic strengthening and anxiety-like behaviors after stress exposure. These data suggest that circuit-specific impairment in 2-AG signaling could facilitate functional coupling between the BLA and plPFC and the translation of environmental stress to affective pathology.
Copyright © 2019 Elsevier Inc. All rights reserved.
Combination therapies consisting of multiple short therapeutic RNAs, such as small interfering RNA (siRNA) and microRNA (miRNA), have enormous potential in cancer treatment as they can precisely silence a specific set of oncogenes and target multiple disease-related pathways. However, clinical use of siRNA/miRNA combinations is limited by the availability of safe and efficient systemic delivery systems with sufficient tumor penetrating and endosomal escaping capabilities. This study reports on the development of multifunctional tumor-penetrating mesoporous silica nanoparticles (iMSNs) for simultaneous delivery of siRNA (siPlk1) and miRNA (miR-200c), using encapsulation of a photosensitizer indocyanine green (ICG) to facilitate endosomal escape and surface conjugation of the iRGD peptide to enable deep tumor penetration. Increased cell uptake of the nanoparticles was observed in both 3D tumor spheroids in vitro and in orthotopic MDA-MB-231 breast tumors in vivo. Using a galectin-8 recruitment assay, we showed that reactive oxygen species generated by ICG upon light irradiation functioned as an endosomolytic stimulus that caused release of the siRNA/miRNA combination from endosomes. Co-delivery of the therapeutic RNAs displayed combined cell killing activity in cancer cells. Systemic intravenous treatment of metastatic breast cancer with the iMSNs loaded with siPlk1 and miR-200c resulted in a significant suppression of the primary tumor growth and in marked reduction of metastasis upon short light irradiation of the primary tumor. This work demonstrates that siRNA-miRNA combination assisted by the photodynamic effect and tumor penetrating delivery system may provide a promising approach for metastatic cancer treatment.
Matsushita et al. describe a model of acute kidney injury to chronic kidney disease progression in mice surviving cardiac arrest: mice develop severe acute kidney injury that initially recovers but is followed by the onset of impaired renal function on longer-term follow-up. These findings suggest that distinct cardiorenal toxicities and/or injury dynamics are operative in this cardiac arrest model that do not occur in traditional models of acute kidney injury, providing new opportunities for therapeutic and biomarker discovery for an important clinical problem.
Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.