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Supplementary Figure 1 from Epithelial to Mesenchymal Transition in Human Breast Epithelial Cells Transformed by 17β-Estradiol

The estrogen dependence of breast cancer has long been recognized; however, the role of 17β-estradiol (E2) in cancer initiation was not known until we showed that it induces complete neoplastic transformation of the human breast epithelial cells MCF-10F. E2 treatment of MCF-10F cells progressively induced high colony efficiency and loss of ductulogenesis in early transformed (trMCF) cells and invasiveness in Matrigel invasion chambers. The cells that crossed the chamber membrane were collected and identified as bsMCF; their subclones were designated bcMCF; and the cells harvested from carcinoma formation in severe combined immunodeficient mice were designated caMCF. These phenotypes correlated with gene dysregulation during the progression of the transformation. The highest number of dysregulated genes was observed in caMCF, being slightly lower in bcMCF, and lowest in trMCF. This order was consistent with the extent of chromosome aberrations (caMCF > bcMCF >>> trMCF). Chromosomal amplifications were found in 1p36.12-pter, 5q21.1-qter, and 13q21.31-qter. Losses of the complete chromosome 4 and 8p11.21-23.1 were found only in tumorigenic cells. In tumor-derived cell lines, additional losses were found in 3p12.1-14.1, 9p22.1-pter, and 18q11.21-qter. Functional profiling of dysregulated genes revealed progressive changes in the integrin signaling pathway, inhibition of apoptosis, acquisition of tumorigenic cell surface markers, and epithelial-mesenchymal transition. In tumorigenic cells, the levels of E-cadherin, epithelial membrane antigen, and various keratins were low and CD44E/CD24 were negative, whereas SNAI2, vimentin, S100A4, FN1, HRAS, transforming growth factor β1, and CD44H were high. The phenotypic and genomic changes triggered by estrogen exposure that lead normal cells to tumorigenesis confirm the role of this steroid hormone in cancer initiation. [Cancer Res 2007;67(23):11147–57]

Supplementary Figure 2 from Epithelial to Mesenchymal Transition in Human Breast Epithelial Cells Transformed by 17β-Estradiol

Supplementary Figure 3 from Epithelial to Mesenchymal Transition in Human Breast Epithelial Cells Transformed by 17β-Estradiol

Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial–mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

To compare computer-based 3D-analysis for quantification of the femorotibial joint space width (JSW) using weight-bearing cone beam CT (WB-CT), non-weight-bearing multi-detector CT (NWB-CT), and weight-bearing conventional radiographs (WB-XR).Twenty-six participants prospectively underwent NWB-CT, WB-CT, and WB-XR of the knee. For WB-CT and NWB-CT, the average and minimal JSW was quantified by 3D-analysis of the minimal distance of any point of the subchondral tibial bone surface and the femur. Associations with mechanical leg axes and osteoarthritis were evaluated. Minimal JSW of WB-CT was further compared to WB-XR. Two-tailed p-values of0.05 were considered significant.Significant differences existed of the average medial and lateral JSW between WB-CT and NWB-CT (medial: 4.7 vs 5.1 mm [P = 0.028], lateral: 6.3 vs 6.8 mm [P = 0.008]). The minimal JSW on WB-XR (medial:3.1 mm, lateral:5.8 mm) were significantly wider compared to WB-CT and NWB-CT (both medial:1.8 mm, lateral:2.9 mm, all p 0.001), but not significantly different between WB-CT and NWB-CT (all p ≥ 0.869). Significant differences between WB-CT and NWB-CT existed in participants with varus knee alignment for the average and the minimal medial JSW (p = 0.004 and p = 0.011) and for participants with valgus alignment for the average lateral JSW (p = 0.013). On WB-CT, 25% of the femorotibial compartments showed bone-on-bone apposition, which was significantly higher when compared to NWB-CT (10%,P = 0.008) and WB-XR (8%,P = 0.012).Combining WB-CT with 3D-based assessment allows detailed quantification of the femorotibial joint space and the effect of knee alignment on JSW. WB-CT demonstrates significantly more bone-on-bone appositions, which are underestimated or even undetectable on NWB-CT and WB-XR.

Objective The oblique orientation of the cervical neural foramina challenges the implementation of a short MRI protocol with concurrent excellent visualization of the spine. While sagittal oblique T2-weighted sequences permit good evaluation of the cervical neuroforamina, all segments may not be equally well depicted on a single sequence and conspicuity of foraminal stenosis may be limited. 3D T2-weighted sequences can be reformatted in arbitrary planes, including the sagittal oblique. We set out to compare 3D T2w SPACE sequences with sagittal oblique reformations and sagittal oblique 2D T2w TSE sequences for the evaluation of cervical foraminal visibility and stenosis. Materials and methods Sixty consecutive patients who underwent MRI of the cervical spine with sagittal oblique 2D T2w TSE and 3D T2w SPACE sequences were included. Image homogeneity of the sequences was evaluated. Imaging sets were assessed for structure visibility and foraminal stenosis by two independent readers. Results of the sequences were compared by Wilcoxon matched-pairs tests. Interreader agreement was evaluated by weighted κ. Results Visibility of most structures was rated good to excellent on both sequences (mean visibility scores ≥ 4.5 of 5), though neuroforaminal contents were better seen on sagittal oblique T2w TSE (mean scores 4.1–4.6 vs. 3.1–4.1 on 3D T2w SPACE, p Conclusion 3D T2w SPACE is comparable with sagittal oblique 2D T2w TSE in the evaluation of cervical neural foramina.

Activation of the transcription factor, the aryl hydrocarbon receptor (AHR), in normal human epidermal keratinocytes (NHEKs) increased AHR binding in the promoters of the glucose transporter, SLC2A1, and the glycolytic enzyme, enolase 1 (ENO1). This increased chromatin binding corresponded with AHR-dependent decreases in levels of SLC2A1 and ENO1 mRNA, protein and activities. Studies of the ENO1 promoter showed activation of the AHR decreases the transcription of ENO1. Glycolysis was lowered by activation of the AHR as measured by decreases in glucose uptake and the production of pyruvate and lactate. Levels of ATP were also decreased. Down-regulation of glucose metabolism, either by activation of the AHR, inhibition of glycolysis, inhibition of glucose transport, or inhibition of enolase, increased SIRT1 protein levels in NHEKs and the immortalized keratinocyte cell line, N/TERT-1. This increase in SIRT1 was abrogated by the addition of exogenous pyruvate. Moreover, keratinocyte differentiation in response to downregulation of glycolysis, either by activation of the AHR, inhibition of glucose transport, or inhibition of enolase, was dependent on SIRT1. These results indicate that regulation of glycolysis controls keratinocyte differentiation, and that activation of the AHR, by lowering the expression of SLC2A1 and ENO1, can determine this fate.

To determine the cutaneous effects of in utero and lactational exposure to the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), pregnant C57BL/6J mice were exposed by gavage to a vehicle or 5 μg TCDD/kg body weight at embryonic day 12 and epidermal barrier formation and function were studied in their offspring from postnatal day 1 (P1) through adulthood. TCDD-exposed pups were born with acanthosis. This effect was AHR-dependent and subsided by P6 with no evidence of subsequent inflammatory dermatitis. The challenge of adult mice with MC903 showed similar inflammatory responses in control and treated animals, indicating no long-term immunosuppression to this chemical. Chloracne-like sebaceous gland hypoplasia and cyst formation were observed in TCDD-exposed P21 mice, with concomitant microbiome dysbiosis. These effects were reversed by P35. CYP1A1 and CYP1B1 expression in the skin was increased in the exposed mice until P21, then declined. Both CYP proteins co-localized with LRIG1-expressing progenitor cells at the infundibulum. CYP1B1 protein also co-localized with a second stem cell niche in the isthmus. These results indicate that this exposure to TCDD causes a chloracne-like effect without inflammation. Transient activation of the AhR, due to the shorter half-life of TCDD in mice, likely contributes to the reversibility of these effects.

SUMMARYThe epidermis forms a barrier that defends the body from desiccation and entry of harmful substances, while sensing and integrating environmental signals. The tightly orchestrated cellular changes required for the proper formation and maintenance of this epidermal barrier occur in the context of the skin microbiome. Using germ free mice, we demonstrate the microbiota is necessary for proper differentiation and repair of the epidermal barrier. These effects were mediated by the aryl hydrocarbon receptor (AHR) in keratinocytes, a xenobiotic receptor also implicated in epidermal differentiation. Murine skin lacking keratinocyte AHR was more susceptible to barrier damage and infection, during steady state and epicutaneous sensitization. Colonization with a defined consortium of human skin isolates restored barrier competence in an AHR-dependent manner. We reveal a fundamental mechanism whereby the microbiota regulates skin barrier formation and repair, with far-reaching implications for the numerous skin disorders characterized by epidermal barrier dysfunction.

Activation of the aryl hydrocarbon receptor (AHR) in normal human epidermal keratinocytes (NHEKs) accelerates keratinocyte terminal differentiation through metabolic reprogramming and reactive oxygen species (ROS) production. Of the three NOS isoforms, NOS3 is significantly increased at both the RNA and protein levels by exposure to the very potent and selective ligand of the AHR, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Inhibition of NOS with the chemical N-nitro-l-arginine methyl ester (l-NAME) reversed TCDD-induced cornified envelope formation, an endpoint of terminal differentiation, as well as the expression of filaggrin (FLG), a marker of differentiation. Conversely, exposure to the NO-donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), increased the number of cornified envelopes above control levels and augmented the levels of cornified envelopes formed in response to TCDD treatment and increased the expression of FLG. This indicates that nitric oxide signaling can increase keratinocyte differentiation and that it is involved in the AHR-mediated acceleration of differentiation. As the nitrosylation of cysteines is a mechanism by which NO affects the structure and functions of proteins, the S-nitrosylation biotin switch technique was used to measure protein S-nitrosylation. Activation of the AHR increased the S-nitrosylation of two detected proteins of about 72 and 20 kD in size. These results provide new insights into the role of NO and protein nitrosylation in the process of epithelial cell differentiation, suggesting a role of NOS in metabolic reprogramming and the regulation of epithelial cell fate.

Gelenkprothesen und andere orthopädische Implantate werden bei vielen Patienten mit muskuloskelettalen Erkrankungen eingesetzt. Während diese Operationen häufig ein gutes klinisches Ergebnis zeigen, sind im Verlauf bei vielen Patienten radiologische Untersuchungen notwendig. Orthopädische Implantate führen jedoch in der MRT und der CT zu starken Metallartefakten. Dieser Artikel stellt mehrere grundlegende Methoden sowie fortgeschrittene Techniken zur Reduktion dieser Artefakte für MRT und CT vor, um eine diagnostische Untersuchung bei Patienten mit Metallimplantaten zu ermöglichen. MRT und CT werden so zu wichtigen und zuverlässigen Modalitäten, um Patienten mit Gelenkprothesen und orthopädischen Implantaten zu untersuchen.

The original version of this article, published on 14 May 2020, unfortunately contained a mistake.

Objective To assess whether preserved dorsal and ventral midsagittal tissue bridges after traumatic cervical spinal cord injury (SCI) encode tract-specific electrophysiologic properties and are predictive of appropriate recovery. Methods In this longitudinal study, we retrospectively assessed MRI scans at 1 month after SCI that provided data on width and location (dorsal vs ventral) of midsagittal tissue bridges in 28 tetraplegic patients. Regression analysis assessed associations between midsagittal tissue bridges and motor- and sensory-specific electrophysiologic recordings and appropriate outcome measures at 12 months after SCI. Results Greater width of dorsal midsagittal tissue bridges at 1 month after SCI identified patients who were classified as being sensory incomplete at 12 months after SCI (p = 0.025), had shorter sensory evoked potential (SEP) latencies (r = −0.57, p = 0.016), and had greater SEP amplitudes (r = 0.61, p = 0.001). Greater width of dorsal tissue bridges predicted better light-touch score at 12 months (r = 0.40, p = 0.045) independently of baseline clinical score and ventral tissue bridges. Greater width of ventral midsagittal tissue bridges at 1 month identified patients who were classified as being motor incomplete at 12 months (p = 0.002), revealed shorter motor evoked potential (MEP) latencies (r = −0.54, p = 0.044), and had greater ratios of MEP amplitude to compound muscle action potential amplitude (r = 0.56, p = 0.005). Greater width of ventral tissue bridges predicted better lower extremity motor scores at 12 months (r = 0.41, p = 0.035) independently of baseline clinical score and dorsal tissue bridges. Conclusion Midsagittal tissue bridges, detectable early after SCI, underwrite tract-specific electrophysiologic communication and are predictors of appropriate sensorimotor recovery. Neuroimaging biomarkers of midsagittal tissue bridges may be integrated into the diagnostic workup, prediction of recovery, and patients' stratification in clinical trials.

Zusammenfassung: Metallartefakte erschweren die Beurteilung von Computer- (CT) und Magnetresonanztomographie (MRT) bei Patienten mit Hüftprothesen. Neuere technische Entwicklungen erlauben heute, beide Methoden bei Patienten mit schmerzhaften Hüftprothesen mit Erfolg einzusetzen. Neuere Technologien zur Metallartefaktreduktion in der CT sind unter anderem iterative Rekonstruktionsalgorithmen und Dual-Energy-CT, in der MRT neuere Sequenzen wie SEMAC ("slice-encoding for metal artifact correction") oder MAVRIC ("multi-acquisition variable-resonance image combination"). Lockerung der Hüftprothese, Osteolysen durch Abriebpartikel und Pseudotumoren bei Metall-Metall-Prothesen sind wichtige Krankheitsbilder bei Hüftprothesen. Infektionen, Frakturen, Tendinopathien, Sehnenrupturen, Muskel- und Nervenschäden sowie heterotope Ossifikationen sind weitere Ursachen einer schmerzhaften Hüftprothese.

The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

A novel pathway of vitamin D activation by CYP11A has previously been elucidated. To define the mechanism of action of its major dihydroxy-products, we tested the divergence and overlap between the gene expression profiles of human epidermal keratinocytes treated with either CYP11A1-derived 20,23(OH)2D3 or classical 1,25(OH)2D3. Both secosteroids have significant chemical similarity with the only differences being the positions of the hydroxyl groups. mRNA was isolated and examined by microarray analysis using Illumina&rsquo, s HumanWG-6 chip/arrays and subsequent bioinformatics analyses. Marked differences in the up- and downregulated genes were observed between 1,25(OH)2D3- and 20,23(OH)2D3-treated cells. Hierarchical clustering identified both distinct, opposite and common (overlapping) gene expression patterns. CYP24A1 was a common gene strongly activated by both compounds, a finding confirmed by qPCR. Ingenuity pathway analysis identified VDR/RXR signaling as the top canonical pathway induced by 1,25(OH)2D3. In contrast, the top canonical pathway induced by 20,23(OH)2D3 was AhR, with VDR/RXR being the second nuclear receptor signaling pathway identified. QPCR analyses validated the former finding by revealing that 20,23(OH)2D3 stimulated CYP1A1 and CYP1B1 gene expression, effects located downstream of AhR. Similar stimulation was observed with 20(OH)D3, the precursor to 20,23(OH)2D3, as well as with its downstream metabolite, 17,20,23(OH)3D3. Using a Human AhR Reporter Assay System we showed marked activation of AhR activity by 20,23(OH)2D3, with weaker stimulation by 20(OH)D3. Finally, molecular modeling using an AhR LBD model predicted vitamin D3 hydroxyderivatives to be good ligands for this receptor. Thus, our microarray, qPCR, functional studies and molecular modeling indicate that AhR is the major receptor target for 20,23(OH)2D3, opening an exciting area of investigation on the interaction of different vitamin D3-hydroxyderivatives with AhR and the subsequent downstream activation of signal transduction pathways in a cell-type-dependent manner.

Status epilepticus (SE) is a life-threatening state of persisting or repetitive seizure activity with often permanent altered level of consciousness. Despite its high morbidity and mortality there is no consensus about the best strategy to treat specific forms of SE. The compromise between the danger related to untreated and persistent seizure activity and the possible damage induced by unnecessary aggressive treatments is challenging. Knowledge about the determinants and reliable prediction models of outcome early in the course of SE is fundamental for rapid treatment modulation and for planning the level of monitoring. This review compiles the current evidence for outcome prediction based on clinical determinants in adult SE patients

The purpose of the study was to retrospectively compare three-dimensional gadolinium-enhanced magnetic resonance angiography (conventional MRA) with MRA accelerated by a parallel acquisition technique (fast MRA) for the assessment of visceral arteries, using 64-detector-row computed tomography angiography (MDCTA) as the reference standard. Eighteen patients underwent fast MRA (imaging time 17s), conventional MRA (29s) and MDCTA of the abdomen and pelvis. Two independent readers assessed subjective image quality and the presence of arterial stenosis. Data were analysed on per-patient and per-segment bases. Fast MRA yielded better subjective image quality in all segments compared with conventional MRA (P = 0.012 for reader 1, P = 0.055 for reader 2) because of fewer motion-induced artefacts. Sensitivity and specificity of fast MRA for the detection of arterial stenosis were 100% for both readers. Sensitivity of conventional MRA was 89% for both readers, and specificity was 100% (reader 1) and 99% (reader 2). Differences in sensitivity between the two types of MRA were not significant for either reader. Interobserver agreement for the detection of arterial stenosis was excellent for fast (κ = 1.00) and good for conventional MRA (κ = 0.76). Thus, subjective image quality of visceral arteries remains good on fast MRA compared with conventional MRA, and the two techniques do not differ substantially in the grading of arterial stenosis, despite the markedly reduced acquisition time of fast MRA

Like many other animals, our behavior often follows a familiar pattern each day. We tend to wake up with the morning sunlight and start by eating breakfast to satisfy our hunger. Then, at night, most of us sleep, and our bodies use chemical building blocks from the day's meals to replenish and repair our tissues. As such, its not just our behavior that shows a daily cycle. The countless chemical reactions that keep us alive, collectively referred to as our metabolism, also change over the course of each day. Daily patterns of activity, known as circadian rhythms, can be seen even at the level of individual cells. Each cell in the body has its own molecular clock that works by rhythmically cycling the levels of different molecules. Proteins called CLOCK and BMAL1 trigger the production of proteins PER and CRY. As levels of PER and CRY rise, they interfere with CLOCK and BMAL1, essentially switching off their own production. Then, levels of PER and CRY fall and the cycle starts again. Thus, these molecules rise and fall throughout the day to drive circadian rhythms, similar to how a pendulum swings back and forth to keep a clock ticking. Metabolism and circadian rhythms are clearly linked, but it is not well understood how this works. Now, Wible, Ramanathan et al. identify a protein called NRF2 as an important bridge between the molecular clock and metabolism. NRF2 is a activated by hydrogen peroxide, a byproduct of cell metabolism, and when activated this protein grabs hold of DNA to increase the activity of specific genes. A combination of experiments revealed that mouse liver cells need NRF2 to maintain a normal circadian rhythm, and a closer inspection of the liver cells revealed that NRF2 specifically attaches to part of the gene for a clock protein called CRY2. This enhances the production of this protein, which in turn, switches CLOCK and BMAL1 off. In this way, NRF2 links metabolism signals to the ticking of the circadian clock. Previous research has shown a link between shift work, which disrupt these rhythms, and metabolic diseases like obesity and diabetes. Understanding more about the underlying molecular biology that links metabolism to circadian rhythms could help scientists to find new ways to improve public health.

Antea Group and KULeuven were awarded a project in Flanders to identify the regions exporting high sediment loads to unnavigable watercourses and the sedimentation zones within them. Two types of models are applied: hydrological sediment export models (SEM) and hydraulic sediment transport models (STM). The influence of erosion control measures on sediment export as well as river engineering measures needs to be taken into account. A concept will be developed to connect the SEM and STM, enabling the sediment to be routed from upstream to the sedimentation zones. Results of the study will be used by the Flemish government to plan erosion control measures, estimate future sedimentation volumes, steer sedimentation and optimize river engineering and dredging works. Finally, model results could also be used to obtain better insights to the re-suspension risks of contaminated sediment in watercourses.

Continuous video-EEG is recommended for patients with altered consciousness; as compared to routine EEG (lasting

The Kelch-like erythroid-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) signaling pathway is the subject of several clinical trials evaluating the effects of Nrf2 activation on the prevention of cancer and diabetes and the treatment of chronic kidney disease and multiple sclerosis. 3H-1,2-dithiole-3-thione (D3T) and 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) are representative members of two distinct series of Nrf2 chemical activators. Previous reports have described activator-specific effects on Nrf2-dependent gene regulation and physiologic outcomes. Here we used a robust chemical genomics approach to characterize expression profiles between D3T and CDDO-Im in livers from wild-type and Nrf2-null mice. At equally efficacious doses in wild-type mice, 406 genes show common RNA responses to both treatments. These genes enriched the Nrf2-regulated pathways of antioxidant defense and xenobiotic metabolism. In addition, 197 and 745 genes were regulated uniquely in response to either D3T or CDDO-Im, respectively. Functional analysis of the D3T-regulated set showed a significant enrichment of Nrf2-regulated enzymes involved in cholesterol biosynthesis. This result was supported by Nrf2-dependent increases in lanosterol synthase and CYP51 protein expression. CDDO-Im had no effect on cholesterol biosynthesis regardless of the dose tested. However, unlike D3T, CDDO-Im resulted in Nrf2-dependent elevation of peroxisome proliferator α and Kruppel-like factor 13, as well as the coactivator peroxisome proliferator γ coactivator 1β, together indicating regulation of β-oxidation and lipid metabolic pathways. These findings provide novel insights into the pharmacodynamic action of these two activators of Keap1-Nrf2 signaling. Although both compounds modify Keap1 to affect canonical cytoprotective gene expression, additional unique sets of Nrf2-dependent genes were regulated by each agent with enrichment of selective metabolic pathways.

On the basis of the authors' experience with 20 patients, CO2 laser resection of palatal tumours has proved to be a good alternative to conventional surgical resection. The CO2 laser beam permits precise resection, due to only slight intra-operative bleeding coupled with use of the operating microscope. Wound healing is good and post-operative pain remarkably little

Among emerging non- albicans Candida species, Candida parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatments for such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting susceptibility to azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate, including upregulation of ergosterol biosynthesis pathway genes ERG2 , ERG5 , ERG6 , ERG11 , ERG24 , ERG25 , and UPC2 . Whole-genome sequencing revealed that the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate's allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins.

Background: Development of the epidermal permeability barrier (EPB) is essential for neonatal life. Defects in this barrier are found in many skin diseases such as atopic dermatitis. Objective: We investigated the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development and function of the EPB. Methods: Timed-pregnant C57BL/6J mice were gavaged with corn oil or TCDD (10 μg/kg body weight) on gestation day 12. Embryos were harvested on embryonic day (E) 15, E16, E17, and postnatal day (PND) 1. Results: A skin permeability assay showed that TCDD accelerated the development of the EPB, beginning at E15. This was accompanied by a significant decrease in transepidermal water loss (TEWL), enhanced stratification, and formation of the stratum corneum (SC). The levels of several ceramides were significantly increased at E15 and E16. PND1 histology revealed TCDD-induced acanthosis and epidermal hyperkeratosis. This was accompanied by disrupted epidermal tight junction (TJ) function, with increased dye leakage at the terminal claudin-1–staining TJs of the stratum granulosum. Because the animals did not have enhanced rates of TEWL, a commonly observed phenotype in animals with TJ defects, we performed tape-stripping. Removal of most of the SC resulted in a significant increase in TEWL in TCDD-exposed PND1 pups compared with their control group. Conclusions: These findings demonstrate that in utero exposure to TCDD accelerates the formation of an abnormal EPB with leaky TJs, warranting further study of environmental exposures, epithelial TJ integrity, and atopic disease. Citation: Muenyi CS, Leon Carrion S, Jones LA, Kennedy LH, Slominski AT, Sutter CH, Sutter TR. 2014. Effects of in utero exposure of C57BL/6J mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin on epidermal permeability barrier development and function. Environ Health Perspect 122:1052–1058; http://dx.doi.org/10.1289/ehp.1308045

Multiple studies show that molecular genetic changes and epigenetic modifications affect the risk of cognitive disability or impairment. However, the role of epigenetic variation in cognitive development of neurotypical young children remains largely unknown. Using data from a prospective, community-based study of mother-infant pairs, we investigated the association of DNA methylation patterns in neonatal umbilical cord blood with cognitive and language development at 1 year of age. No CpG loci achieved genome-wide significance, although a small number of weakly suggestive associations with Bayley-III Receptive Communication scales were noted. While umbilical cord blood is a convenient resource for genetic analyses of birth outcomes, our results do not provide conclusive evidence that its use for DNA methylation profiling yields epigenetic markers that are directly related to postnatal neurocognitive outcomes at 1 year of age.

Background: We previously introduced a method based on post hoc pairwise comparisons to analyze gene expression responses. This method utilized directed graphs to represent gene response to all treatment pairs. It has been found useful in identifying structure-activity relationships among drugs and differentiating genes sharing similar functional pathways. Directed graphs are descriptive, visually expressive and can benefit subsequent functional analysis. Results: mDAG is a web-based software package based on this established method for the analysis, visualization, and interpretation of patterns of responses in gene expression data involving multiple treatments. Genes with the same directed graph patterns hypothetically share similar biological function, which may be further analyzed using external tools. To facilitate subsequent functional analysis, several well-known tools have been incorporated into mDAG to help users explore hypotheses about gene function and regulation. This tool is useful for any studies that analyze comparatively response patterns in gene expression data with multiple treatments (chemicals, cell types, etc.). Availability: The (server/personal/demo) software is freely available at http://cetus.cs. memphis.edu/mdag.

Chloracne is commonly observed in humans exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); yet, the mechanism of toxicity is not well understood. Using normal human epidermal keratinocytes, we investigated the mechanism of TCDD-mediated enhancement of epidermal differentiation by integrating functional genomic, metabolomic, and biochemical analyses. TCDD increased the expression of 40% of the genes of the epidermal differentiation complex found on chromosome 1q21 and 75% of the genes required for de novo ceramide biosynthesis. Lipid analysis demonstrated that eight of the nine classes of ceramides were increased by TCDD, altering the ratio of ceramides to free fatty acids. TCDD decreased the expression of the glucose transporter, SLC2A1, and most of the glycolytic transcripts, followed by decreases in glycolytic intermediates, including pyruvate. NADH and Krebs cycle intermediates were decreased, whereas NAD(+) was increased. Mitochondrial glutathione (GSH) reductase activity and the GSH/glutathione disulfide ratio were decreased by TCDD, ultimately leading to mitochondrial dysfunction, characterized by decreased inner mitochondrial membrane potential and ATP production, and increased production of the reactive oxygen species (ROS), hydrogen peroxide. Aryl hydrocarbon receptor (AHR) antagonists blocked the response of many transcripts to TCDD, and the endpoints of decreased ATP production and differentiation, suggesting regulation by the AHR. Cotreatment of cells with chemical antioxidants or the enzyme catalase blocked the TCDD-mediated acceleration of keratinocyte cornified envelope formation, an endpoint of terminal differentiation. Thus, TCDD-mediated ROS production is a critical step in the mechanism of this chemical to accelerate keratinocyte differentiation.

RL95-2 endometrial cancer cells were used to study cytochrome P450-mediated chemopreventative mechanisms of four flavonoids found in foods. To investigate enzymatic CYP1 inhibition, intact cells were induced with benzo(a)pyrene or 2,3,7,8-tetrachlorodibenzo- p -dioxin. Quercetin, kaempferol and myricetin inhibited CYP1 activity dose-dependently with IC 50 s ranging from 2.2 to 4 μM; while amentoflavone was inactive. Further experiments were designed to determine if flavonoids also interacted with the AhR or caused a decrease in CYP1 protein or mRNA expression. CYP1A1 protein expression was inhibited in cells co-treated with TCDD and quercetin, kaempferol or myricetin compared with TCDD alone, but amentoflavone was ineffective. Relatively higher (∼7-fold) basal levels of CYP1B1 protein were not significantly affected by flavonoid treatments. In general, at the message level significant inhibition of induced CYP1A1 or CYP1B1 was not detected following flavonoid cotreatment. Despite the common inhibitory effects of quercetin, kaempferol, and myricetin on induced CYP1A1-dependent activity and protein expression, the mechanisms of CYP1 inhibition in this cell line are complex and dependent on the CYP gene, AhR inducer and the flavonoid.

In this methods article, we describe collection and storage of clinically acquired blood and adipose samples for transcript analysis in an ongoing study exploring obesity in renal transplant recipients. Total ribonucleic acid (RNA) was isolated from whole blood using the LeukoLOCK™ Total RNA Isolation System (n = 4), and comparisons between fresh and frozen samples were made. Abdominal subcutaneous adipose samples (n = 4) were obtained during kidney transplantation, flash frozen, and stored at −80°C. Adipose RNA was extracted using either the STAT-60 method modified for lipids or Trizol plus RNeasy extraction. Affymetrix HG-U133 plus 2.0 arrays and Affymetrix Human Gene 1.0 ST arrays were used for both blood and adipose transcriptome analysis. Purity, quality, and quantity of RNA were high with comparable results using both array platforms. © 2011 Wiley Periodicals, Inc. Res Nurs Health 34:408–418, 2011

Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1 . In cultures of normal human epidermal keratinocytes dioxin accelerates cell differentiation, as measured by the formation of cornified envelopes. We show that this acceleration is mediated by the AHR; also, that dioxin increases the expression of several genes known to be regulated by ARNT, which have critical roles in the cornification and epidermal barrier function of the skin. Importantly, we demonstrate that all of these responses are opposed by ligand-activation of the EGF receptor (R), an important regulator of keratinocyte cell fate. In the CYP1A1 enhancer, EGFR activation prevents recruitment of the p300 coactivator, although not affecting the binding of the AHR or ARNT. The total cellular level of p300 protein does not decrease, and overexpression of p300 relieves EGFR-mediated repression of transcription, indicating that p300 is a critical target for the repression of the AHR complex by EGFR signaling. These results provide a mechanism by which 2,3,7,8-tetrachlorodibenzo- p -dioxin is able to disrupt epidermal homeostasis and identify EGFR signaling as a regulator of the AHR. This signaling may modulate the incidence and severity of chloracne and be of therapeutic relevance to human poisonings by dioxin.

To better understand the mechanisms involved in aluminum toxicity and tolerance in plants, microarray technology was used to evaluate changes in gene expression in Arabidopsis thaliana under Al stress. With the use of Affymetrix Arabidopsis ATH1 Genechip, a comparison of RNA expression profiles was made between control and Al-treated Arabidopsis seedlings. A total of 256 genes were identified as Al-responsive. Ninety-four genes were shown to be up-regulated and 162 were down-regulated; comprising 1.1 % of the 24 000 Arabidopsis genes. Real-time RT-PCR was used to confirm the microarray data. The analysis showed that a large number of transcription factors and several putative signaling components were up-regulated by aluminum. Chloroplast structural and photosynthetic genes were, in general, down-regulated. A number of previously identified Al-responsive genes, e.g. GST, Auxin-regulated, Peroxidase, and Chitinase, were up-regulated by Al-stress, whereas Wali 3 and Wali 4 were down-regulated. We also identified several up-regulated genes involved in vacuolar signaling, sorting and docking. Three genes were also up-regulated by Al-stress, Ras GTP-binding protein, ABC-cassette binding, and the AtELP1 receptor genes, have previously been documented as responsive to drought and/or oxidative stress and may play important roles the detoxification of Al ions by transportation and storage into root vacuoles. Ultrastructural changes in the roots tips cells of Arabidopsis were evaluated using transmission electron microscopy and energy-dispersive X-ray analysis with scanning electron microscopy and results showed Al accumulation in the root tips of Arabidopsis.

The reduction of the aflatoxin B 1 (AFB 1) dialdehyde metabolite to its corresponding mono and dialcohols, catalyzed by aflatoxin B 1-aldehyde reductase (AFAR, rat AKR7A1, and human AKR7A3), is greatly increased in livers of rats treated with numerous chemoprotective agents. Recombinant human AKR7A3 has been shown to reduce the AFB 1-dialdehyde at rates greater than those of the rat AKR7A1. The activity of AKR7A1 or AKR7A3 may detoxify the AFB 1-dialdehyde, which reacts with proteins, and thereby inhibits AFB 1-induced toxicity; however, direct experimental evidence of this hypothesis was lacking. Two human B lymphoblastoid cell lines, designated pMF6/1A2/AKR7A1 and pMF6/1A2, were genetically engineered to stably express AKR7A1 and/or cytochrome P4501A2 (1A2). The pMF6/1A2/AKR7A1 cells were refractory to the cytotoxic effects of 3 ng/mL AFB 1, in comparison to pM6/1A2 cells, which were more sensitive. Diminished protection occurred at higher concentrations of AFB 1 in pMF6/1A2/AKR7A1 cells, suggesting that additional factors were influencing cell survival. COS-7 cells were transfected with either vector control, rat AKR7A1, or human AKR7A3, and the cells were treated with AFB 1-dialdehyde. There was a 6-fold increase in the dialdehyde LC 50, from 66 microM in vector-transfected cells to 400 microM in AKR7A1-transfected cells, and an 8.5-fold increase from 35 microM in vector-transfected cells to 300 microM in AKR7A3-transfected cells. In both cases, this protective effect of the AFAR enzyme was accompanied by a marked decrease in protein adducts. Fractionation of the cellular protein showed that the mitochondria/nuclei and microsomal fractions contained the highest concentration of protein adducts. The levels of human AKR7A3 and AKR7A2 were measured in 12 human liver samples. The expression of AKR7A3 was detectable in all livers and lower than those of AKR7A2 in 11 of the 12 samples. Overall, these results provide the first direct evidence of a role for rat AKR7A1 and human AKR7A3 in protection against AFB 1-induced cytotoxicity and protein adduct formation.

The estrogen dependence of breast cancer has long been recognized; however, the role of 17β-estradiol (E2) in cancer initiation was not known until we showed that it induces complete neoplastic transformation of the human breast epithelial cells MCF-10F. E2 treatment of MCF-10F cells progressively induced high colony efficiency and loss of ductulogenesis in early transformed (trMCF) cells and invasiveness in Matrigel invasion chambers. The cells that crossed the chamber membrane were collected and identified as bsMCF; their subclones were designated bcMCF; and the cells harvested from carcinoma formation in severe combined immunodeficient mice were designated caMCF. These phenotypes correlated with gene dysregulation during the progression of the transformation. The highest number of dysregulated genes was observed in caMCF, being slightly lower in bcMCF, and lowest in trMCF. This order was consistent with the extent of chromosome aberrations (caMCF > bcMCF >>> trMCF). Chromosomal amplifications were found in 1p36.12-pter, 5q21.1-qter, and 13q21.31-qter. Losses of the complete chromosome 4 and 8p11.21-23.1 were found only in tumorigenic cells. In tumor-derived cell lines, additional losses were found in 3p12.1-14.1, 9p22.1-pter, and 18q11.21-qter. Functional profiling of dysregulated genes revealed progressive changes in the integrin signaling pathway, inhibition of apoptosis, acquisition of tumorigenic cell surface markers, and epithelial-mesenchymal transition. In tumorigenic cells, the levels of E-cadherin, epithelial membrane antigen, and various keratins were low and CD44E/CD24 were negative, whereas SNAI2, vimentin, S100A4, FN1, HRAS, transforming growth factor β1, and CD44H were high. The phenotypic and genomic changes triggered by estrogen exposure that lead normal cells to tumorigenesis confirm the role of this steroid hormone in cancer initiation. [Cancer Res 2007;67(23):11147–57]