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IMPACT STATEMENT - Subarachnoid hemorrhage (SAH) is associated with vasospasm that is refractory to traditional vasodilators, and inhibition of vasospasm after SAH remains a large unmet clinical need. SAH causes changes in the phosphorylation state of the small heat shock proteins (HSPs), HSP20 and HSP27, in the vasospastic vessels. In this study, the levels of HSP27 and HSP20 were manipulated using nanotechnology to mimic the intracellular phenotype of SAH-induced vasospasm, and the effect of this manipulation was tested on vasomotor responses in intact tissues. This work provides insight into potential therapeutic targets for the development of more effective treatments for SAH induced vasospasm.
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.
Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.
Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.
Pregnancy produces important health-related needs, and expectant families have turned to technologies to meet them. The ability to predict needs and technology preferences might aid in connecting families with resources. This study examined the relationships among Multidimensional Health Locus of Control (MHLC) scores, information-seeking behaviors, and health-related needs in 71 pregnant women and 29 caregivers. Internal MHLC scores were positively correlated with information-seeking behaviors, including website and patient portal use. Higher Chance scores were associated with decreased portal or pregnancy website use (p=0.002), with the exception of FitPregnancy.com (p=0.02). MHLC scores were not significantly correlated with number of health-related needs or whether needs were met. Individuals with needs about disease management had higher Powerful Others scores (p=0.01); those with questions about tests had lower Powerful Others scores (p=0.008). MHLC scores might be used to identify individuals less likely to seek information and to predict need types.
Immediately following a fracture, a fibrin laden hematoma is formed to prevent bleeding and infection. Subsequently, the organized removal of fibrin, via the protease plasmin, is essential to permit fracture repair through angiogenesis and ossification. Yet, when plasmin activity is lost, the depletion of fibrin alone is insufficient to fully restore fracture repair, suggesting the existence of additional plasmin targets important for fracture repair. Previously, activated matrix metalloproteinase 9 (MMP-9) was demonstrated to function in fracture repair by promoting angiogenesis. Given that MMP-9 is a defined plasmin target, it was hypothesized that pro-MMP-9, following plasmin activation, promotes fracture repair. This hypothesis was tested in a fixed murine femur fracture model with serial assessment of fracture healing. Contrary to previous findings, a complete loss of MMP-9 failed to affect fracture healing and union through 28 days post injury. Therefore, these results demonstrated that MMP-9 is dispensable for timely fracture union and cartilage transition to bone in fixed femur fractures. Pro-MMP-9 is therefore not a significant target of plasmin in fracture repair and future studies assessing additional plasmin targets associated with angiogenesis are warranted.
Bacterial DNA has been reported in the placenta and amniotic fluid by several independent groups of investigators. However, it's taxonomic overlap with fetal and maternal bacterial DNA in different sites has been poorly characterized. Here, we determined the presence of bacterial DNA in the intestines and placentas of fetal mice at gestational day 17 (n = 13). These were compared to newborn intestines (n = 15), maternal sites (mouth, n = 6; vagina, n = 6; colon, n = 7; feces, n = 8), and negative controls to rule out contamination. The V4 region of the bacterial 16S rRNA gene indicated a pattern of bacterial DNA in fetal intestine similar to placenta but with higher phylogenetic diversity than placenta or newborn intestine. Firmicutes were the most frequently assignable phylum. SourceTracker analysis suggested the placenta as the most commonly identifiable origin for fetal bacterial DNA, but also over 75% of fetal gut genera overlapped with maternal oral and vaginal taxa but not with maternal or newborn feces. These data provide evidence for the presence of bacterial DNA in the mouse fetus.
Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein σ1, which projects from the virion surface. The σ1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from β-spiral repeats; and a globular head with receptor-binding properties. The σ1 tail is predicted to form an α-helical coiled coil. Although σ1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of σ1 molecules released from virions identified three regions of flexibility, including one at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential σ1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) σ1 tail domains. Both molecules feature extended α-helical coiled coils, with T1L σ1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered σ1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for σ1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus σ1 and provide insight into the stabilization of a multidomain virus attachment protein. While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry of nonenveloped viruses. The filamentous reovirus attachment protein σ1 binds cellular receptors; contains regions of predicted flexibility, including one at the fiber midpoint; and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large σ1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the σ1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus σ1 attachment protein.
Copyright © 2018 American Society for Microbiology.
Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.