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Incidences of invasive pulmonary aspergillosis, an infection caused predominantly by Aspergillus fumigatus, have increased due to the growing number of immunocompromised individuals. While A. fumigatus is reliant upon deficiencies in the host to facilitate invasive disease, the distinct mechanisms that govern the host-pathogen interaction remain enigmatic, particularly in the context of distinct immune modulating therapies. To gain insights into these mechanisms, RNA-Seq technology was utilized to sequence RNA derived from lungs of 2 clinically relevant, but immunologically distinct murine models of IPA on days 2 and 3 post inoculation when infection is established and active disease present. Our findings identify notable differences in host gene expression between the chemotherapeutic and steroid models at the interface of immunity and metabolism. RT-qPCR verified model specific and nonspecific expression of 23 immune-associated genes. Deep sequencing facilitated identification of highly expressed fungal genes. We utilized sequence similarity and gene expression to categorize the A. fumigatus putative in vivo secretome. RT-qPCR suggests model specific gene expression for nine putative fungal secreted proteins. Our analysis identifies contrasting responses by the host and fungus from day 2 to 3 between the two models. These differences may help tailor the identification, development, and deployment of host- and/or fungal-targeted therapeutics.

Lead (Pb) is a prominent toxic metal in natural and engineered systems. Current knowledge on Pb toxicity to the activated sludge has been limited to short-term (≤24 h) toxicity. The effect of extended Pb exposure on process performance, bacterial viability, and community compositions remains unknown. We quantified the 24-h and 7-day Pb toxicity to chemical oxygen demand (COD) and NH3–N removal, bacterial viability, and community compositions using lab-scale experiments. Our results showed that 7-day toxicity was significantly higher than the short-term 24-h toxicity. Ammonia-oxidizing bacteria were more susceptible than the heterotrophs to Pb toxicity. The specific oxygen uptake rate responded quickly to Pb addition and could serve as a rapid indicator for detecting Pb pollutions. Microbial viability decreased linearly with the amount of added Pb at extended exposure. The bacterial community diversity was markedly reduced with elevated Pb concentrations. Surface analysis suggested that the adsorbed form of Pb could have contributed to its toxicity along with the dissolved form. Our study provides for the first time a systematic investigation of the effect of extended exposure of Pb on the performance and microbiology of aerobic treatment processes, and it indicates that long-term Pb toxicity has been underappreciated by previous studies.

Embryonic eyelid closure involves forward movement and ultimate fusion of the upper and lower eyelids, an essential step of mammalian ocular surface development. Although its underlying mechanism of action is not fully understood, a functional mitogen-activated protein kinase kinase kinase 1 (MAP3K1) is required for eyelid closure. Here we investigate the molecular signatures of MAP3K1 in eyelid morphogenesis. At mouse gestational day E15.5, the developmental stage immediately prior to eyelid closure, MAP3K1 expression is predominant in the eyelid leading edge (LE) and the inner eyelid (IE) epithelium. We used laser capture microdissection (LCM) to obtain highly enriched LE and IE cells from wild type and MAP3K1-deficient fetuses and analyzed genome-wide expression profiles. The gene expression data led to the identification of three distinct developmental features of MAP3K1. First, MAP3K1 modulated Wnt and Sonic hedgehog signals, actin reorganization, and proliferation only in LE but not in IE epithelium, illustrating the temporal-spatial specificity of MAP3K1 in embryogenesis. Second, MAP3K1 potentiated AP-2α expression and SRF and AP-1 activity, but its target genes were enriched for binding motifs of AP-2α and SRF, and not AP-1, suggesting the existence of novel MAP3K1-AP-2α/SRF modules in gene regulation. Third, MAP3K1 displayed variable effects on expression of lineage specific genes in the LE and IE epithelium, revealing potential roles of MAP3K1 in differentiation and lineage specification. Using LCM and expression array, our studies have uncovered novel molecular signatures of MAP3K1 in embryonic eyelid closure., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The mechanisms for provisioning maternal resources to offspring in placental mammals involve complex interactions between maternally regulated and fetally regulated gene networks in the placenta, a tissue that is derived from the zygote and therefore of fetal origin. Here we describe a novel use of an embryo transfer system in mice to identify gene networks in the placenta that are regulated by the mother. Mouse embryos from the same strain of inbred mice were transferred into a surrogate mother either of the same strain or from a different strain, allowing maternal and fetal effects on the placenta to be separated. After correction for sex and litter size, maternal strain overrode fetal strain as the key determinant of fetal weight (P < 0.0001). Computational filtering of the placental transcriptome revealed a group of 81 genes whose expression was solely dependent on the maternal strain [P < 0.05, false discovery rate (FDR) < 0.10]. Network analysis of this group of genes yielded highest statistical significance for pathways involved in the regulation of cell growth (such as insulin-like growth factors) as well as those involved in regulating lipid metabolism [such as the low-density lipoprotein receptor-related protein 1 (LRP1), LDL, and HDL], both of which are known to play a role in fetal development. This novel technique may be generally applied to identify regulatory networks involved in maternal-fetal interaction and eventually help identify molecular targets in disorders of fetal growth.

Background: The vertebrate aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular responses to environmental polycyclic and halogenated compounds. The naive receptor is believed to reside in an inactive cytosolic complex that translocates to the nucleus and induces transcription of xenobiotic detoxification genes after activation by ligand., Objectives: We conducted an integrative genomewide analysis of AHR gene targets in mouse hepatoma cells and determined whether AHR regulatory functions may take place in the absence of an exogenous ligand., Methods: The network of AHR-binding targets in the mouse genome was mapped through a multipronged approach involving chromatin immunoprecipitation/chip and global gene expression signatures. The findings were integrated into a prior functional knowledge base from Gene Ontology, interaction networks, Kyoto Encyclopedia of Genes and Genomes pathways, sequence motif analysis, and literature molecular concepts., Results: We found the naive receptor in unstimulated cells bound to an extensive array of gene clusters with functions in regulation of gene expression, differentiation, and pattern specification, connecting multiple morphogenetic and developmental programs. Activation by the ligand displaced the receptor from some of these targets toward sites in the promoters of xenobiotic metabolism genes., Conclusions: The vertebrate AHR appears to possess unsuspected regulatory functions that may be potential targets of environmental injury.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic effects of its xenobiotic ligands and acts as an environmental checkpoint during the cell cycle. We expressed stably integrated, Tet-Off-regulated AHR variants in fibroblasts from AHR-null mice to further investigate the AHR role in cell cycle regulation. Ahr+/+ fibroblasts proliferated significantly faster than Ahr-/- fibroblasts did, and exposure to a prototypical AHR ligand or deletion of the ligand-binding domain did not change their proliferation rates, indicating that the AHR function in cell cycle was ligand independent. Growth-promoting genes, such as cyclin and cyclin-dependent kinase genes, were significantly down-regulated in Ahr-/- cells, whereas growth-arresting genes, such as the transforming growth factor beta1 (TGF-beta1) gene, extracellular matrix (ECM)-related genes, and cyclin-dependent kinase inhibitor genes, were up-regulated. Ahr-/- fibroblasts secreted significantly more TGF-beta1 into the culture medium than Ahr+/+ fibroblasts did, and Ahr-/- showed increased levels of activated Smad4 and TGF-beta1 mRNA. Inhibition of TGF-beta1 signaling by overexpression of Smad7 reversed the proliferative and gene expression phenotype of Ahr-/- fibroblasts. Changes in TGF-beta1 mRNA accumulation were due to stabilization resulting from decreased activity of TTP, the tristetraprolin RNA-binding protein responsible for mRNA destabilization through AU-rich motifs. These results show that the Ah receptor possesses interconnected intrinsic cellular functions, such as ECM formation, cell cycle control, and TGF-beta1 regulation, that are independent of activation by either exogenous or endogenous ligands and that may play a crucial role during tumorigenesis.

The aryl hydrocarbon receptor (AHR) mounts the body's main molecular defense against environmental toxicants by inducing a battery of genes encoding xenobiotic metabolizing proteins. The AHR is activated by polycyclic aromatic hydrocarbon toxicants, including the pervasive teratogen and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). The TCDD-activated AHR significantly changes the cytoplasmic mRNA levels of hundreds of genes, but little is known of the mechanism by which the activated AHR causes such a strong effect on global gene expression. We used high-density microarrays to compare nuclear and cytoplasmic RNA levels from untreated and TCDD-treated mouse embryonic fibroblasts (MEF) to test the hypotheses that (1) TCDD has a large impact on nuclear RNA levels and (2) that cytoplasmic RNA levels are dependent on nuclear RNA levels. We found that nuclear RNA levels are strongly affected by TCDD, and that nuclear and cytoplasmic RNA levels are only weakly correlated, indicating that other regulatory mechanisms are controlling cytoplasmic RNA levels. The nuclear RNAs most affected by TCDD encode proteins involved in nuclear RNA processing and transcription. We conclude that although the AHR regulates key xenobiotic metabolizing genes at the transcriptional level, a larger impact of the TCDD-activated AHR may be at post-transcriptional levels.

The recent sequencing of a large number of Xenopus tropicalis expressed sequences has allowed development of a high-throughput approach to study Xenopus global RNA gene expression. We examined the global gene expression similarities and differences between the historically significant Xenopus laevis model system and the increasingly used X.tropicalis model system and assessed whether an X.tropicalis microarray platform can be used for X.laevis. These closely related species were also used to investigate a more general question: is there an association between mRNA sequence divergence and differences in gene expression levels? We carried out a comprehensive comparison of global gene expression profiles using microarrays of different tissues and developmental stages of X.laevis and X.tropicalis. We (i) show that the X.tropicalis probes provide an efficacious microarray platform for X.laevis, (ii) describe methods to compare interspecies mRNA profiles that correct differences in hybridization efficiency and (iii) show independently of hybridization bias that as mRNA sequence divergence increases between X.laevis and X.tropicalis differences in mRNA expression levels also increase.

Dual-channel long oligonucleotide microarrays are in widespread use. Although much attention has been given to proper experimental design and analysis regarding long oligonucleotide microarrays, relatively little information is available concerning the optimization of protocols. We carried out a series of microarray experiments designed to investigate the effects of different levels of target concentration and hybridization times using a long oligonucleotide library. Based on principles developed from nucleic acid renaturation kinetics studies, we show that increasing the time of hybridization from 18 h to 42 h and 66 h, especially when lower than optimal concentrations of target were used, significantly improved the quality of the microarray results. Longer hybridization times significantly increased the number of spots detected, signal-to-noise ratios, and the number of differentially expressed genes and correlations among replicate arrays. We conclude that at 18 h of incubation, target-to-probe hybridization has not reached equilibrium and that a relatively high proportion of nonspecific hybridization occurs. This result is striking, given that most, if not all, published microarray protocols stipulate 8-24 h for hybridization. Using shorter than optimal hybridization times (i.e., not allowing hybridization to reach equilibrium) has the consequence of underestimating the fold change of differentially expressed genes and of missing less represented sequences.

The AP-1 transcription factor plays a critical role in regulating tumor cell proliferation and has been implicated in controlling a program of gene expression that mediates cell motility and invasion in vitro. We have utilized two dominant negative AP-1 constructs, TAM67 and aFos, each fused to GFP, to investigate the role of AP-1 complexes in an invasive, clinically derived human tumor cell line, HT-1080. As expected, high levels of both GFP-TAM67 and GFP-aFos arrested HT-1080 cells in the G1 phase of the cell cycle. Strikingly, at low levels GFP-aFos, but not GFP-TAM67, caused a change in colony morphology, impairment of directional motility in a monolayer wound healing assay, as well as inhibition of chemotaxis and haptotaxis. Microarray analysis identified a novel set of AP-1 target genes, including the tumor suppressor TSCL-1 and regulators of actin cytoskeletal dynamics, including the gelsolin-like actin capping protein CapG. The demonstration that AP-1 regulates the expression of genes involved in tumor cell motility and cytoskeletal dynamics in a clinically derived human tumor cell line identifies new pathways of control for tumor cell motility.

The molecular basis for the adverse biological effects of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD), a pervasive environmental toxin, is largely unknown. TCDD is a ligand for the cytosolic aromatic hydrocarbon receptor (AHR) which mediates the transcriptional induction of the xenobiotic metabolizing genes in the CYP1 family of cytochromes P450. Previous studies have suggested that the AHR may carry out important functions in the cell in addition to metabolizing toxins. We present gene expression profiles of smooth muscle cells from wild type and Ahr(-/-) mice that show significant changes in the RNA levels of the transforming growth factor-beta3 (Tgfb3) gene and genes involved in the modulation and processing of TGF-beta. The RNA expression profiles support a hypothesis that in the wild type, the AHR represses Tgfb gene expression and affects the gene expression of several TGF-beta-modulating and processing genes. We also observed that RNA levels increased for TGF-beta2, CYP1b1, and TGF-beta-related genes in Ahr(-/-) smooth muscle cells exposed to TCDD. These data are consistent with a hypothesis that TCDD stimulates the TGF-beta2 signaling pathway in the absence of the AHR to activate the Cyp1b1 gene. The above results provide a possible explanation for some of the multiple biological effects of TCDD and the physiological role played by the AHR in the absence of environmental agents.

Benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands for the aryl hydrocarbon receptor (AHR). High-density oligonucleotide microarrays were used to generate global gene expression profiles of wild-type and Ahr(-/-) vascular smooth muscle cells (SMCs) from mouse aorta. To determine whether there are signaling pathways other than the AHR involved in B[a]P metabolism, wild-type and AHR knockout (Ahr(-/-) SMCs were exposed to B[a]P. Two signaling pathways, represented by TGF-beta2 and IGF-1, were identified as potential candidates of an AHR alternate pathway for cells to respond to B[a]P. The wild-type SMCs responded similarly to B[a]P and TCDD in the regulation of a small set of common genes known to respond to the activated AHR (e.g., glutamine S-transferase). However, wild-type SMCs responded in a way that involves many additional genes, suggesting that a very divergent cellular response may be involved when SMCs are exposed to the two classic inducers of the AHR. In contrast, many more genes in the Ahr(-/-) cells responded similarly to B[a]P and TCDD, including Cyp1b1, than responded differently, which indicates that eliminating the AHR is effective for investigating potential alternate cellular mechanisms that respond to B[a]P and TCDD.

Neurofibromatosis type 1 (NF1) patients are predisposed to learning disabilities, macrocephaly, and brain tumors as well as abnormalities on magnetic resonance imaging that are postulated to result from abnormal myelination. Here we show that Nf1+/- spinal cords in adult mice have more than twofold-increased numbers of NG2+ progenitor cells. Nf1-/- embryonic spinal cords have increased numbers of Olig2+ progenitors. Also, cultures from Nf1 mutant embryos with hemizygous and biallelic Nf1 mutations have dramatically increased numbers of CNS oligodendrocyte progenitor cells. In medium that allows growth of neuroepithelial cells and glial progenitors, mutant cells hyper-respond to FGF2, have increased basal and FGF-stimulated Ras-GTP, and fail to accumulate when treated with a farnesyltransferase inhibitor. Cell accumulation results in part from increased proliferation and decreased cell death. In contrast to wild-type cells, Nf1-/- progenitors express the glial differentiation marker O4 while retaining expression of the progenitor marker nestin. Nf1 mutant progenitors also abnormally coexpress the glial differentiation markers O4 and GFAP. Importantly, Nf1-/- spinal cord-derived oligodendrocyte progenitors, which are amplified 12-fold, retain the ability to form oligodendrocytes after in vivo transplantation. The data reveal a key role for neurofibromin and Ras signaling in the maintenance of CNS progenitor cell pools and also suggest a potential role for progenitor cell defects in the CNS abnormalities of NF1 patients.

We describe a key role for the CD44 transmembrane glycoprotein in Schwann cell-neuron interactions. CD44 proteins have been implicated in cell adhesion and in the presentation of growth factors to high affinity receptors. We observed high CD44 expression in early rat neonatal nerves at times when Schwann cells proliferate but low expression in adult nerves, where CD44 was found in some nonmyelinating Schwann cells and to varying extents in some myelinating fibers. CD44 constitutively associated with erbB2 and erbB3, receptor tyrosine kinases that heterodimerize and signal in Schwann cells in response to neuregulins. Moreover, CD44 significantly enhanced neuregulin-induced erbB2 phosphorylation and erbB2-erbB3 heterodimerization. Reduction of CD44 expression in vitro resulted in loss of Schwann cell-neurite adhesion and Schwann cell apoptosis. CD44 is therefore crucial for maintaining neuron-Schwann cell interactions at least partly by facilitating neuregulin-induced erbB2-erbB3 activation.

Cultured Schwann cells secreted low levels (30 pg/ml/1.5 x 10(6) cells) of a 45-kDa neuregulin protein and showed constitutive activation of a neuregulin receptor, Erb-B3, suggesting the existence of an autocrine loop involving neuregulins in Schwann cells. RT-PCR analyses indicated that Schwann cells and fibroblasts in culture produced SMDF/n-ARIA and NDF but not GGF neuregulin messages. Schwann cell and fibroblast neuregulin messages encoded both beta and alpha domains; Schwann cell transcripts encoded only transmembrane neuregulin forms while fibroblast messages encoded transmembrane and secreted forms. SMDF/n-ARIA and NDF messages were also expressed in early postnatal rat sciatic nerve, suggesting a role for neuregulins in peripheral nerve development. An anti-neuregulin antibody inhibited the mitogenic response of Schwann cells to cultured neurons and to extracts of cultured neurons or embryonic brain, consistent with the accepted paracrine role of neuregulins on Schwann cells. Surprisingly, the same antibody inhibited Schwann cell proliferation stimulated by several unrelated mitogens including bFGF, HGF, and TGF-beta1. These data implicate both paracrine and autocrine pathways involving neuregulin form(s) in Schwann cell mitogenic responses., (Copyright 1997 Academic Press.)