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Copper toxicity and copper limitation can both be effective host defense mechanisms against pathogens. Tolerance of high copper by fungi makes toxicity as a defense mechanism largely ineffective against fungal pathogens. A forward genetic screen for Histoplasma capsulatum mutant yeasts unable to replicate within macrophages showed the Ctr3 copper transporter is required for intramacrophage proliferation. Ctr3 mediates copper uptake and is required for growth in low copper. Transcription of the CTR3 gene is induced by differentiation of H. capsulatum into pathogenic yeasts and by low available copper, but not decreased iron. Low expression of a CTR3 transcriptional reporter by intracellular yeasts implies that phagosomes of non-activated macrophages have moderate copper levels. This is further supported by the replication of Ctr3-deficient yeasts within the phagosome of non-activated macrophages. However, IFN-γ activation of phagocytes causes restriction of phagosomal copper as shown by upregulation of the CTR3 transcriptional reporter and by the failure of Ctr3-deficient yeasts, but not Ctr3 expressing yeasts, to proliferate within these macrophages. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during phases of the innate immune response but are attenuated after the onset of adaptive immunity. Thus, while technical limitations prevent direct measurement of phagosomal copper concentrations and copper-independent factors can influence gene expression, both the CTR3 promoter induction and the attenuation of Ctr3-deficient yeasts indicate activation of macrophages switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition.

Human cytomegalovirus (HCMV) infects up to 80% of the world’s population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hr after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon 6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMV’s established roles in these processes.

Copper toxicity and copper limitation can both be effective host defense mechanisms against pathogens. Tolerance of high copper by fungi makes toxicity as a defense mechanism largely ineffective against fungal pathogens. A forward genetic screen for Histoplasma capsulatum mutant yeasts unable to replicate within macrophages showed the Ctr3 copper transporter is required for intramacrophage proliferation. Ctr3 mediates copper uptake and is required for growth in low copper. Transcription of the CTR3 gene is induced by differentiation of H. capsulatum into pathogenic yeasts and by low available copper, but not decreased iron. Low expression of a CTR3 transcriptional reporter by intracellular yeasts implies that phagosomes of non-activated macrophages have moderate copper levels. This is further supported by the replication of Ctr3-deficient yeasts within the phagosome of non-activated macrophages. However, IFN-[gamma] activation of phagocytes causes restriction of phagosomal copper as shown by upregulation of the CTR3 transcriptional reporter and by the failure of Ctr3-deficient yeasts, but not Ctr3 expressing yeasts, to proliferate within these macrophages. Accordingly, in a respiratory model of histoplasmosis, Ctr3-deficient yeasts are fully virulent during phases of the innate immune response but are attenuated after the onset of adaptive immunity. Thus, while technical limitations prevent direct measurement of phagosomal copper concentrations and copper-independent factors can influence gene expression, both the CTR3 promoter induction and the attenuation of Ctr3-deficient yeasts indicate activation of macrophages switches the phagosome from a copper-replete to a copper-depleted environment, forcing H. capsulatum reliance on Ctr3 for copper acquisition.

Human cytomegalovirus (HCMV) is a β-herpesvirus which is ubiquitous in the human population. HCMV has the largest genome of all known human herpesviruses, and thus encodes a large array of proteins that affect pathogenesis in different cell types. Given the large genome and the ability of HCMV to replicate in a range of cells, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded in the HCMV genome that are homologous to human G protein-coupled receptors (GPCRs); these viral-encoded GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In the current study, we find that deletion of all four vGPCR genes from a clinical isolate of HCMV severely attenuates lytic replication in both primary human salivary gland epithelial cells, as well as ARPE-19 retinal epithelial cells as evidenced by significant decreases in immediate early gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of immediate early, early, and late gene expression, as well as reduced viral production. We find that similar to US28, UL33 constitutively activates Gαq-dependent PLC-β signaling to high levels in these epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5' UTRs to create novel UL33 proteins that are all capable of constitutive Gαq signaling. Taken together, these studies suggest that UL33 driven signaling is important for lytic HCMV replication in cells of epithelial origin.

Human cytomegalovirus (HCMV) infects up to 80% of the world’s population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hr after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon 6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMV’s established roles in these processes.

Human cytomegalovirus (HCMV) infects up to 80% of the world's population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hours after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of exon-6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMV's established roles in these processes.

Human cytomegalovirus (HCMV) is a highly successful pathogen which is prevalent throughout the world and estimated to infect up to 80% of the human population globally. HCMV causes lifelong persistent infections which can severely impact the health of those who are immunocompromised and lead to increased risk of developing other diseases for even the immunocompetent. Epithelial cells make up numerous tissues and organ systems throughout the body and represent a biologically important cell type for HCMV infection; however, epithelial models are limited and HCMV pathogenesis is most extensively studied in the context of retinal epithelial infection. The salivary glands are an important site for HCMV replication as the virus establishes a persistent infection in acinar or ductal epithelial cells which then transmit infectious virus to new hosts via saliva. This contrasts with other tissue systems which are marked by an acute infection, followed by clearance of the virus through host immune system function. We explored HCMV epithelial tropism in these studies by use of a primary human salivary gland-derived epithelial (hSGE) cell system. During infection of hSGE cells we observed that, in contrast to the established epithelial model, HCMV can persistently infect these cells by use of self-limiting lytic replication. This self-limiting of infection is likely one of many mechanisms used by the virus to avoid immune detection and subsequent clearance. We additionally identified that epithelial defective strains of HCMV can be greatly enhanced for epithelial infection in hSGE and other epithelial models by use of histone deacetylase inhibitors. These inhibitors were clearly identified to be capable of overcoming the loss of the Pentamer, which is an essential viral protein for replication in epithelial cells. This Pentamer is described as being an entry mediator by unclear mechanisms. Our data reveals that virions lacking Pentamer can be rescued post-entry and recontextualizes our overall understanding of how the Pentamer functions to promote epithelial cell infection. Based on mouse models with the related murine cytomegalovirus we examined a role for virally encoded G protein coupled receptors (vGPCR) in the context of hSGE infection. Using a battery of vGPCR expressing HCMV mutants, we identified that vGPCRs are required to first initiate lytic replication in hSGE cells and then establishment of persistent infection. We discovered that of the 4 vGPCRs which HCMV encodes, UL33, UL78, and US28 are the 3 essential vGPCRs for these phenotypes. By characterizing mutants expressing 2 of those 3 vGPCRs we identified there are redundant functions for initiation of lytic replication but different functions for establishing persistent infection. These studies expand on our understanding of which virulence factors are important for lytic replication in epithelial cells by HCMV. These studies further identify distinct differences in HCMV replication between a retinal and salivary model of epithelial infection, underscoring the importance of accounting for cellular tropism of HCMV in future studies.

The InSight lander will deliver geophysical instruments to Mars in 2018, including seismometers installed directly on the surface (Seismic Experiment for Interior Structure, SEIS). Routine operations will be split into two services, the Mars Structure Service (MSS) and Marsquake Service (MQS), which will be responsible, respectively, for defining the structure models and seismicity catalogs from the mission. The MSS will deliver a series of products before the landing, during the operations, and finally to the Planetary Data System (PDS) archive. Prior to the mission, we assembled a suite of a priori models of Mars, based on estimates of bulk composition and thermal profiles. Initial models during the mission will rely on modeling surface waves and impact-generated body waves independent of prior knowledge of structure. Later modeling will include simultaneous inversion of seismic observations for source and structural parameters. We use Bayesian inversion techniques to obtain robust probability distribution functions of interior structure parameters. Shallow structure will be characterized using the hammering of the heatflow probe mole, as well as measurements of surface wave ellipticity. Crustal scale structure will be constrained by measurements of receiver function and broadband Rayleigh wave ellipticity measurements. Core interacting body wave phases should be observable above modeled martian noise levels, allowing us to constrain deep structure. Normal modes of Mars should also be observable and can be used to estimate the globally averaged 1D structure, while combination with results from the InSight radio science mission and orbital observations will allow for constraint of deeper structure.

To the Editor: In their article (April 8 issue), 1 Carlson and Link state that because of surgical risks — in particular, risks to the facial nerve — the practice of intentionally leaving a tumor remnant has gained popularity. Such an approach is usually considered in the case of large vestibular schwannomas but not for small or medium-sized tumors, for which radiosurgery is the treatment of choice (supported by level II evidence), 2 since it offers a high degree of facial and cochlear functional preservation. 3 For large vestibular schwannomas, a “nerve-centered approach” with planned intracapsular resection offers equivalent functional outcomes 4 but may . . .