Endocrine disruptors of sex hormone activities.

Sex hormones, such as androgens, estrogens and progestins are naturally occurring compounds that tightly regulate endocrine systems in a variety of living organisms. Uncontrolled environmental exposure to these hormones or their biological and synthetic mimetics has been widely documented. Furthermore, water contaminants penetrate soil to affect flora, fauna and ultimately humans. Because endocrine systems evolved to respond to very small changes in hormone levels, the low levels found in the environment cannot be ignored. The combined actions of sex hormones with glucocorticoids and other nuclear receptors disruptors creates additional level of complexity including the newly described "dynamic assisted loading" mechanism. We reviewed the extensive literature pertaining to world-wide detection of these disruptors and created a detailed Table on the development and current status of methods used for their analysis.

A Novel Combination Therapy Using Rosuvastatin and Lactobacillus Combats Dextran Sodium Sulfate-Induced Colitis in High-Fat Diet-Fed Rats by Targeting the TXNIP/NLRP3 Interaction and Influencing Gut Microbiome Composition.

Inflammasome targeting and controlling dysbiosis are promising therapeutic approaches to control ulcerative colitis. This report is the first to investigate the mechanisms underlying the coloprotective effects of rosuvastatin and Lactobacillus and their combined therapy on dextran sodium sulfate (DSS)-induced colitis in high-fat diet (HFD)-fed rats. Our results demonstrate the aggravation of intestinal inflammation as a consequence of an HFD following DSS administration. An association between dyslipidemia, LDL oxidation, CD36 expression, ROS generation, thioredoxin-interacting protein (TXNIP) upregulation, and NLRP3 inflammasome activation was demonstrated by DSS exposure in HFD-fed rats. We demonstrated that rosuvastatin/Lactobacillus significantly suppressed the DSS/HFD-induced increase in colon weight/length ratio, DAI, MDI, and myeloperoxidase, as well as corrected dysbiosis and improved histological characteristics. Additionally, caspase-1 activity and IL-1ß-driven pyroptotic activity was significantly reduced. Rosuvastatin/Lactobacillus showed prominent anti-inflammatory effects as revealed by the IL-10/IL-12 ratio and the levels of TNF-α and IL-6. These latter effects may be attributed to the inhibition of phosphorylation-induced activation of NF-κB and a concomitant reduction in the expression of NLRP3, pro-IL-1ß, and pro-IL-18. Furthermore, rosuvastatin/Lactobacillus reduced Ox-LDL-induced TXNIP and attenuated the inflammatory response by inhibiting NLRP3 inflammasome assembly. To conclude, rosuvastatin/Lactobacillus offers a safe and effective strategy for the management of ulcerative colitis.

Endoscopic-CT: Learning-Based Photometric Reconstruction for Endoscopic Sinus Surgery.

In this work we present a method for dense reconstruction of anatomical structures using white light endoscopic imagery based on a learning process that estimates a mapping between light reflectance and surface geometry. Our method is unique in that few unrealistic assumptions are considered (i.e., we do not assume a Lambertian reflectance model nor do we assume a point light source) and we learn a model on a per-patient basis, thus increasing the accuracy and extensibility to different endoscopic sequences. The proposed method assumes accurate video-CT registration through a combination of Structure-from-Motion (SfM) and Trimmed-ICP, and then uses the registered 3D structure and motion to generate training data with which to learn a multivariate regression of observed pixel values to known 3D surface geometry. We demonstrate with a non-linear regression technique using a neural network towards estimating depth images and surface normal maps, resulting in high-resolution spatial 3D reconstructions to an average error of 0.53mm (on the low side, when anatomy matches the CT precisely) to 1.12mm (on the high side, when the presence of liquids causes scene geometry that is not present in the CT for evaluation). Our results are exhibited on patient data and validated with associated CT scans. In total, we processed 206 total endoscopic images from patient data, where each image yields approximately 1 million reconstructed 3D points per image.

Rendering-Based Video-CT Registration with Physical Constraints for Image-Guided Endoscopic Sinus Surgery.

We present a system for registering the coordinate frame of an endoscope to pre- or intra- operatively acquired CT data based on optimizing the similarity metric between an endoscopic image and an image predicted via rendering of CT. Our method is robust and semi-automatic because it takes account of physical constraints, specifically, collisions between the endoscope and the anatomy, to initialize and constrain the search. The proposed optimization method is based on a stochastic optimization algorithm that evaluates a large number of similarity metric functions in parallel on a graphics processing unit. Images from a cadaver and a patient were used for evaluation. The registration error was 0.83 mm and 1.97 mm for cadaver and patient images respectively. The average registration time for 60 trials was 4.4 seconds. The patient study demonstrated robustness of the proposed algorithm against a moderate anatomical deformation.

Vision-based proximity detection in retinal surgery.

In retinal surgery, surgeons face difficulties such as indirect visualization of surgical targets, physiological tremor, and lack of tactile feedback, which increase the risk of retinal damage caused by incorrect surgical gestures. In this context, intraocular proximity sensing has the potential to overcome current technical limitations and increase surgical safety. In this paper, we present a system for detecting unintentional collisions between surgical tools and the retina using the visual feedback provided by the opthalmic stereo microscope. Using stereo images, proximity between surgical tools and the retinal surface can be detected when their relative stereo disparity is small. For this purpose, we developed a system comprised of two modules. The first is a module for tracking the surgical tool position on both stereo images. The second is a disparity tracking module for estimating a stereo disparity map of the retinal surface. Both modules were specially tailored for coping with the challenging visualization conditions in retinal surgery. The potential clinical value of the proposed method is demonstrated by extensive testing using a silicon phantom eye and recorded rabbit in vivo data.

Δ133p53 is an independent prognostic marker in p53 mutant advanced serous ovarian cancer.

BACKGROUND: We aimed to evaluate the clinical relevance of p53 and p73 isoforms that modulate the function of p53. METHODS: This prospective multicentre study included 154 patients with stage III and IV serous ovarian cancer. A functional yeast-based assay and subsequent sequencing were performed to analyse the p53 mutational status. Expression of p53 and p73 isoforms was determined using RT-qPCR. RESULTS: Δ133p53 expression constituted an independent prognostic marker for recurrence-free (hazard ratio=0.571, P=0.016, 95% CI: 0.362-0.899) and overall survival (hazard ratio=0.365, P=0.004, 95% CI: 0.182-0.731) in patients with p53 mutant ovarian cancer (n=121). High Δ40p53 expression was associated with favourable tumour grading (P=0.037) and improved recurrence-free survival (33.4 vs 19.6 months, P=0.029), but not overall survival (43.1 vs 33.6 months, P=0.139), in patients with p53 wild-type cancer (n=33). Neither the p53 mutational status nor p73 isoform expression possessed prognostic significance in the examined ovarian cancer cases. CONCLUSION: Δ133p53 expression was associated with prognosis in the vast majority of ovarian cancer cases, that is, patients with p53 mutant advanced serous carcinomas. Thus, our findings underline the importance of considering the complex p53 regulatory network.

Augmenting capsule endoscopy diagnosis: a similarity learning approach.

The current procedure for diagnosis of Crohn's disease (CD) from Capsule Endoscopy is a tedious manual process which requires the clinician to visually inspect large video sequences for matching and categorization of diseased areas (lesions). Automated methods for matching and classification can help improve this process by reducing diagnosis time and improving consistency of categorization. In this paper, we propose a novel SVM-based similarity learning method for distinguishing between correct and incorrect matches in Capsule Endoscopy (CE). We also show that this can be used in conjunction with a voting scheme to categorize lesion images. Results show that our methods outperform standard classifiers in discriminating similar from dissimilar lesion images, as well as in lesion categorization. We also show that our methods drastically reduce the complexity (training time) by requiring only one half of the data for training, without compromising the accuracy of the classifier.

Quantum phase transition in a 1D transport model with boson-affected hopping: Luttinger liquid versus charge-density-wave behavior.

We solve a very general two-channel fermion-boson model describing charge transport within some background medium by means of a refined pseudosite density-matrix renormalization group technique. Performing a careful finite-size scaling analysis, we determine the ground-state phase diagram and convincingly prove that the model exhibits a metal-insulator quantum phase transition for the half-filled band case. In order to characterize the metallic and insulating regimes we calculate, besides the local particle densities and fermion-boson correlation functions, the kinetic energy, the charge-structure factor, the Luttinger liquid charge exponent, and the single-particle excitation gap for a one-dimensional infinite system.

AFM imaging of protein movements: histone H2A-H2B release during nucleosome remodeling.

Being able to follow assembly/disassembly reactions of biomolecular complexes directly at the single molecule level would be very useful. Here, we use an AFM technique that can simultaneously obtain topographic images and identify the locations of a specific type of protein within those images to monitor the histone H2A component of nucleosomes acted on by human Swi-Snf, an ATP-dependent nucleosome remodeling complex. Activation of remodeling results in significant H2A release from nucleosomes, based on recognition imaging and nucleosome height changes, and changes in the recognition patterns of H2A associated directly with hSwi-Snf complexes.

Expression and regulation of CReMM, a chromodomain helicase-DNA-binding (CHD), in marrow stroma derived osteoprogenitors.

This study follows the expression of CReMM, a new CHD family member, in osteoprogenitors. CReMM expression was analyzed in primary cultured mesnchymal cells from rat and human. Analysis in ex vivo cultured marrow stromal cells (MSC) from rats revealed higher level of CReMM in cells from young (3 months), when compared to cells from old (15 months) rats. CReMM level was higher in human MSC then in mature trabecular bone cells (TBC). Within the MSC population, osteogenic clones showed higher levels of CReMM then non-osteogenic ones. We used bone marrow derived osteogenic cell line (MBA-15) to elaborate on the regulation of CReMM expression in correlation with cell proliferation and co-expression with alkaline phosphatase (ALK). CReMM is highly expressed in proliferating cells and is inversely related to expression of ALK. MBA-15 cells were challenged with dexamethasone (Dex) or 17beta-estradiol and quantification of CReMM at the protein (ELISA) and mRNA (RT-PCR) levels had shown that Dex upregulated CReMM levels. Since CReMM is regulated by Dex, we analyzed the interaction of CReMM with the glucocorticoid receptor (GR), which mediates Dex action. Co-immunopercipitation (Co-IP) demonstrated an association between CReMM and GR. In summary, CReMM is a CHD protein expressed by osteoprogenitors, and we suggest it plays a role in mediating transcriptional response to hormones that coordinate osteoblast function.

Using atomic force microscopy to study nucleosome remodeling on individual nucleosomal arrays in situ.

In eukaryotes, genomic processes like transcription, replication, repair, and recombination typically require alterations in nucleosome structure on specific DNA regions to operate. ATP-dependent nucleosome remodeling complexes provide a major mechanism for carrying out such alterations in vivo. To learn more about the action of these important complexes, we have utilized an atomic force microscopy in situ technique that permits comparison of the same individual molecules before and after activation of a particular process, in this case nucleosome remodeling. This direct approach was used to look for changes induced by the action of the human Swi-Snf remodeling complex on individual, single-copy mouse mammary tumor virus promoter nucleosomal arrays. Using this technique, we detect a variety of changes on remodeling. Many of these changes are larger in scale than suggested from previous studies and involve a number of DNA-mediated events, including a preference for the removal of a complete turn (80 basepairs) of nucleosomal DNA. The latter result raises the possibility of an unanticipated mode of human Swi-Snf interaction with the nucleosome, namely via the 11-nm histone surface.

Nucleosomal arrays can be salt-reconstituted on a single-copy MMTV promoter DNA template: their properties differ in several ways from those of comparable 5S concatameric arrays.

Subsaturated nucleosomal arrays were reconstituted on a single-copy MMTV promoter DNA fragment by salt dialysis procedures and studied by atomic force microscopy. Up to an occupation level of approximately eight nucleosomes on this 1900 bp template, salt reconstitution produces nucleosomal arrays which look very similar to comparably loaded 5S rDNA nucleosomal arrays; i.e., nucleosomes are dispersed on the DNA template. Thus, at these occupation levels, the single-copy MMTV template forms arrays suitable for biophysical analyses. A quantitative comparison of the population features of subsaturated MMTV and 5S arrays detects differences between the two: a requirement for higher histone levels to achieve a given level of nucleosome occupation on MMTV templates, indicating that nucleosome loading is thermodynamically less favorable on this template; a preference for pairwise nucleosome occupation of the MMTV (but not the 5S) template at midrange occupation levels; and an enhanced salt stability for nucleosomes on MMTV versus 5S arrays, particularly in the midrange of array occupation. When average occupation levels exceed approximately eight nucleosomes per template, MMTV arrays show a significant level of mainly intramolecular compaction; 5S arrays do not. Taken together, these results show clearly that the nature of the underlying DNA template can affect the physical properties of nucleosomal arrays. DNA sequence-directed differences in the physical properties of chromatin may have important consequences for functional processes such as gene regulation.

Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser.

One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor-photobleaching methods for FRET have mainly used wide-field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser-scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF-Receptor-Associated-Factors or TRAFs) that are known to homo- and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non-bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser-scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.

Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter.

Activation of gene transcription involves chromatin remodeling by coactivator proteins that are recruited by DNA-bound transcription factors. Local modification of chromatin structure at specific gene promoters by ATP-dependent processes and by posttranslational modifications of histone N-terminal tails provides access to RNA polymerase II and its accompanying transcription initiation complex. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge, low levels of arginine methylation of histones have only recently been documented, and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro and cooperates synergistically with p160-type coactivators (e.g., GRIP1, SRC-1, ACTR) and coactivators with histone acetyltransferase activity (e.g., p300, CBP) to enhance gene activation by steroid and nuclear hormone receptors (NR) in transient transfection assays. In the current study, CARM1 cooperated with GRIP1 to enhance steroid hormone-dependent activation of stably integrated mouse mammary tumor virus (MMTV) promoters, and this coactivator function required the methyltransferase activity of CARM1. Chromatin immunoprecipitation assays and immunofluorescence studies indicated that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process.

A glucocorticoid/retinoic acid receptor chimera that displays cytoplasmic/nuclear translocation in response to retinoic acid. A real time sensing assay for nuclear receptor ligands.

Members of the nuclear receptor superfamily play key roles in a host of physiologic and pathologic processes from embryogenesis to cancer. Some members, including the retinoic acid receptor (RAR), are activated by ligand binding but are unaffected in their subcellular distribution, which is predominantly nuclear. In contrast, several members of the steroid receptor family, including the glucocorticoid receptor, are cytoplasmic and only translocate to the nucleus after ligand binding. We have constructed chimeras between RAR and glucocorticoid receptor that selectively respond to RAR agonists but display cytoplasmic localization in the absence of ligand. These chimeric receptors manifest both nuclear translocation and gene activation functions in response to physiological concentrations of RAR ligands. The ability to achieve regulated subcellular trafficking with a heterologous ligand binding domain has implications both for current models of receptor translocation and for structural-functional conservation of ligand binding domains broadly across the receptor superfamily. When coupled to the green fluorescent protein, chimeric receptors offer a powerful new tool to 1) study mechanisms of steroid receptor translocation, 2) detect dynamic and graded distributions of ligands in complex microenvironments such as embryos, and 3) screen for novel ligands of "orphan" receptors in vivo.

Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter.

We have examined the relationship between transcription and chromatin structure using a tandem array of the mouse mammary tumor virus (MMTV) promoter driving a ras reporter. The array was visualized as a distinctive fluorescent structure in live cells stably transformed with a green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR), which localizes to the repeated MMTV elements after steroid hormone treatment. Also found at the array by immunofluorescence were two different steroid receptor coactivators (SRC1 and CBP) with acetyltransferase activity, a chromatin remodeler (BRG1), and two transcription factors (NFI and AP-2). Within 3 h after hormone addition, arrays visualized by GFP-GR or DNA fluorescent in situ hybridization (FISH) decondensed to varying degrees, in the most pronounced cases from a approximately 0.5-microm spot to form a fiber 1-10 microm long. Arrays later recondensed by 3-8 h of hormone treatment. The degree of decondensation was proportional to the amount of transcript produced by the array as detected by RNA FISH. Decondensation was blocked by two different drugs that inhibit polymerase II, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin. These observations demonstrate a role for polymerase in producing and maintaining decondensed chromatin. They also support fiber-packing models of higher order structure and suggest that transcription from a natural promoter may occur at much higher DNA-packing densities than reported previously.