Iron Regulatory Protein 1 is Required for the Propagation of Inflammation in Inflammatory Bowel Disease.

Objective: Inflammatory bowel diseases (IBD) are complex disorders. Iron accumulates in the inflamed tissue of IBD patients, yet neither a mechanism for the accumulation nor its implication on the course of inflammation are known. We hypothesized that the inflammation modifies iron homeostasis, affects tissue iron distribution and that this in turn perpetuates the inflammation. Design: This study analyzed human biopsies, animal models and cellular systems to decipher the role of iron homeostasis in IBD. Results: We found inflammation-mediated modifications of iron distribution, and iron-decoupled activation of the iron regulatory protein (IRP)1. To understand the role of IRP1 in the course of this inflammation-associated iron pattern, a novel cellular co-culture model was established, that replicated the iron-pattern observed in vivo, and supported involvement of nitric oxide in the activation of IRP1 and the typical iron pattern in inflammation. Importantly, deletion of IRP1 from an IBD mouse model completely abolished both, the misdistribution of iron and intestinal inflammation. Conclusion: These findings suggest that IRP1 plays a central role in the coordination of the inflammatory response in the intestinal mucosa and that it is a viable candidate for therapeutic intervention in IBD.

GECO: gene expression clustering optimization app for non-linear data visualization of patterns.

BACKGROUND: Due to continued advances in sequencing technology, the limitation in understanding biological systems through an "-omics" lens is no longer the generation of data, but the ability to analyze it. Importantly, much of this rich -omics data is publicly available waiting to be further investigated. Although many code-based pipelines exist, there is a lack of user-friendly and accessible applications that enable rapid analysis or visualization of data. RESULTS: GECO (Gene Expression Clustering Optimization; http://www.theGECOapp.com ) is a minimalistic GUI app that utilizes non-linear reduction techniques to rapidly visualize expression trends in many types of biological data matrices (such as bulk RNA-seq or proteomics). The required input is a data matrix with samples and any type of expression level of genes/protein/other with a unique ID. The output is an interactive t-SNE or UMAP analysis that clusters genes (or proteins/other unique IDs) based on their expression patterns across the multiple samples enabling visualization of expression trends. Customizable settings for dimensionality reduction, data normalization, along with visualization parameters including coloring and filters, ensure adaptability to a variety of user uploaded data. CONCLUSION: This local and cloud-hosted web browser app enables investigation of any -omic data matrix in a rapid and code-independent manner. With the continued growth of available -omic data, the ability to quickly evaluate a dataset, including specific genes of interest, is more important than ever. GECO is intended to supplement traditional statistical analysis methods and is particularly useful when visualizing clusters of genes with similar trajectories across many samples (ex: multiple cell types, time course, dose response). Users will be empowered to investigate -omic data with a new lens of visualization and analysis that has the potential to uncover genes of interest, cohorts of co-regulated genes programs, and previously undetected patterns of expression.

Activation of the Wnt/ß-catenin signaling cascade after traumatic nerve injury.

Recent data have shown that preservation of the neuromuscular junction (NMJ) after traumatic nerve injury helps to improve functional recovery with surgical repair via matrix metalloproteinase-3 (MMP3) blockade. As such, we sought to explore additional pathways that may augment this response. Wnt3a has been shown to inhibit acetylcholine receptor (AChR) clustering via ß-catenin-dependent signaling in the development of the NMJ. Therefore, we hypothesized that Wnt3a and ß-catenin are associated with NMJ destabilization following traumatic denervation. A critical size nerve defect was created by excising a 10-mm segment of the sciatic nerve in mice. Denervated muscles were then harvested at multiple time points for immunofluorescence staining, quantitative real-time PCR, and western blot analysis for Wnt3a and ß-catenin levels. Moreover, a novel Wnt/ß-catenin transgenic reporter mouse line was utilized to support our hypothesis of Wnt activation after traumatic nerve injury. The expression of Wnt3a mRNA was significantly increased by 2 weeks post-injury and remained upregulated for 2 months. Additionally, ß-catenin was activated at 2 months post-injury relative to controls. Correspondingly, immunohistochemical analysis of denervated transgenic mouse line TCF/Lef:H2B-GFP muscles demonstrated that the number of GFP-positive cells was increased at the motor endplate band. These collective data support that post-synaptic AChRs destabilize after denervation by a process that involves the Wnt/ß-catenin pathway. As such, this pathway serves as a potential therapeutic target to prevent the motor endplate degeneration that occurs following traumatic nerve injury.

Adalimumab drug and antibody levels as predictors of clinical and laboratory response in patients with Crohn's disease.

BACKGROUND: Adalimumab is an effective treatment for Crohn's disease (CD). Anti-adalimumab antibodies (AAA) and low trough serum drug concentrations have been implicated as pre-disposing factors for treatment failure. AIMS: To assess adalimumab and AAA serum levels, and to examine their association and discriminatory ability with clinical response and serum C-reactive protein (CRP). METHODS: We performed a cross-sectional study using trough sera from adalimumab-treated CD patients. Demographical data, Montreal classification, treatment regimen and clinical status were recorded. Serum adalimumab, AAA and CRP were measured. Receiver operating characteristic analysis and a multivariate regression model were performed to find drug and antibody thresholds for predicting disease activity at time of serum sampling. RESULTS: One hundred and eighteen trough serum samples were included from 71 patients. High adalimumab trough serum concentration was associated with disease remission (Area Under Curve 0.748, P < 0.001). A cut-off drug level of 5.85 µg/mL yielded optimal sensitivity, specificity and positive likelihood ratio for remission prediction (68%, 70.6% and 2.3, respectively). AAA were inversely related with adalimumab drug levels (Spearman's r = -0.411, P < 0.001) and when subdivided into categorical values, positively related with disease activity (P < 0.001). High drug levels and stricturing vs. penetrating or inflammatory phenotype, but not AAA levels, independently predicted disease remission in a multivariate logistic regression model. CONCLUSIONS: Adalimumab drug levels were inversely related to disease activity. High levels of anti-adalimumab antibodies were positively associated with disease activity, but this association was mediated mostly by adalimumab drug levels.

Extended haplotype association study in Crohn's disease identifies a novel, Ashkenazi Jewish-specific missense mutation in the NF-κB pathway gene, HEATR3.

The Ashkenazi Jewish population has a several-fold higher prevalence of Crohn's disease (CD) compared with non-Jewish European ancestry populations and has a unique genetic history. Haplotype association is critical to CD etiology in this population, most notably at NOD2, in which three causal, uncommon and conditionally independent NOD2 variants reside on a shared background haplotype. We present an analysis of extended haplotypes that showed significantly greater association to CD in the Ashkenazi Jewish population compared with a non-Jewish population (145 haplotypes and no haplotypes with P-value <10(-3), respectively). Two haplotype regions, one each on chromosomes 16 and 21, conferred increased disease risk within established CD loci. We performed exome sequencing of 55 Ashkenazi Jewish individuals and follow-up genotyping focused on variants in these two regions. We observed Ashkenazi Jewish-specific nominal association at R755C in TRPM2 on chromosome 21. Within the chromosome 16 region, R642S of HEATR3 and rs9922362 of BRD7 showed genome-wide significance. Expression studies of HEATR3 demonstrated a positive role in NOD2-mediated NF-κB signaling. The BRD7 signal showed conditional dependence with only the downstream rare CD-causal variants in NOD2, but not with the background haplotype; this elaborates NOD2 as a key illustration of synthetic association.

Topological classification and enumeration of RNA structures by genus.

To an RNA pseudoknot structure is naturally associated a topological surface, which has its associated genus, and structures can thus be classified by the genus. Based on earlier work of Harer-Zagier, we compute the generating function Dg,σ (z) = ∑n dg,σ (n)zn for the number dg,σ (n) of those structures of fixed genus g and minimum stack size σ with n nucleotides so that no two consecutive nucleotides are basepaired and show that Dg,σ (z) is algebraic. In particular, we prove that dg,2(n) ∼ kg n3(g−1/2 )γ n2, where γ2 ≈ 1.9685. Thus, for stack size at least two, the genus only enters through the sub-exponential factor, and the slow growth rate compared to the number of RNA molecules implies the existence of neutral networks of distinct molecules with the same structure of any genus. Certain RNA structures called shapes are shown to be in natural one-to-one correspondence with the cells in the Penner-Strebel decomposition of Riemann's moduli space of a surface of genus g with one boundary component, thus providing a link between RNA enumerative problems and the geometry of Riemann's moduli space.

A Wnt kinase network alters nuclear localization of TCF-1 in colon cancer.

Constitutive activation of the Wnt/beta-catenin pathway has been implicated as the primary cause of colon cancer. However, the major transducers of Wnt signaling in the intestine, T-cell factor 1 (TCF-1) and TCF-4, have opposing functions. Knockout of TCF-4 suppresses growth and maintenance of crypt stem cells, whereas knockout of TCF-1 leads to adenomas. These phenotypes suggest that TCF-4 is Wnt-promoting, whereas TCF-1 acts like a tumor suppressor. Our study of TCF expression in human colon crypts reveals a mechanistic basis for this paradox. In normal colon cells, a dominant-negative isoform of TCF-1 (dnTCF-1) is expressed that is equally distributed between nuclear and cytoplasmic compartments. In colon cancer cells, TCF-1 is predominantly cytoplasmic. Localization is because of active nuclear export and is directed by an autocrine-acting Wnt ligand that requires Ca2+/calmodulin-dependent kinase II (CaMKII) activity for secretion and a downstream step in the export pathway. TCF-4 remains nuclear; its unopposed activity is accompanied by downregulation of dnTCF-1 and increased expression of full-length isoforms. Thus, the dnTCF-1 and TCF-4 balance is corrupted in cancer by two mechanisms, a Wnt/CaMKII kinase signal for nuclear export and decreased dnTCF-1 expression. We propose that dnTCF-1 provides homeostatic regulation of Wnt signaling and growth in normal colon, and the alterations in nuclear export and promoter usage contribute to aberrant Wnt activity in colon cancer.

Intestinal adenoma formation and MYC activation are regulated by cooperation between MYB and Wnt signaling.

Aberrant Wnt signaling mediated by mutations affecting APC (adenomatous polyposis coli) or beta-catenin initiates the majority of human colorectal cancers (CRC) and drives tumorigenesis through the activation of specific genes such as MYC. We report here a novel association whereby another oncogenic transcription factor, MYB/c-Myb, is necessary for intestinal adenoma development directed by activated Wnt signaling. APC(Min/+) mice in which c-myb is haploinsufficient survive longer than wild-type APC(Min/+) animals due to a delay in adenoma formation. Intestinal adenomas from APC(Min/+) mice were assessed and found to have high levels of c-myc gene expression. We explored the relationship between activated Wnt signaling and MYB in regulating MYC and found activated beta-catenin in combination with MYB induces robust upregulation of MYC promoter activity, as well as endogenous MYC mRNA and protein expression, in human cells. This cooperation occurred through independent binding of MYB and beta-catenin to the MYC promoter. These data highlight a cooperative function for MYB in the context of activated Wnt signaling and provide a molecular basis for the expression of MYC in CRC.

Exploring prison buprenorphine misuse in the United Kingdom: a qualitative study of former prisoners.

The United Kingdom Ministry of Justice recently highlighted the extent of buprenorphine (Subutex) misuse in English andWelsh prisons, naming it the third most misused drug overall. Yet little is known regarding how illicit buprenorphine is obtained in prison and what influences prisoners to use it. Qualitative research was used to explore prison drug using practices. Thirty men who were former prisoners with a history of injecting drug use were interviewed in depth about their illicit prison drug use, including buprenorphine. Interviews were conducted over 18 months, from August 2006 to January 2008 and were analysed using Framework. The misuse of Subutex by snorting emerged as a significant theme. Accounts suggested that the diversion of prison prescribed Subutex was widespread and prisoners used various tactics to obtain the medication. Various complex and interlinked reasons were given to explain why Subutex was snorted in prison. The main motivation for snorting was to experience a prolonged euphoric opiate effect, believed to help to combat the boredom of being in prison. The price of illicit Subutex in prison was linked to its availability, but it was generally cheaper than heroin, thus contributing to its use. Participants'narratives identified the belief that snorting Subutex in prison was not risk free, but risks were lower than continuing to use other drugs, particularly injecting illicit opiates. The implications of prison Subutex misuse for prisoners, prison medical services, commissioners, and prescribing policy and practice are discussed.

Hepatopancreatic multi-transcript expression patterns in the crayfish Cherax quadricarinatus during the moult cycle.

Alterations of hepatopancreatic multi-transcript expression patterns, related to induced moult cycle, were identified in male Cherax quadricarinatus through cDNA microarray hybridizations of hepatopancreatic transcript populations. Moult was induced by X-organ sinus gland extirpation or by repeated injections of 20-hydroxyecdysone. Manipulated males were sacrificed at premoult or early postmoult, and a reference population was sacrificed at intermoult. Differentially expressed genes among the four combinations of two induction methods and two moult stages were identified. Biologically interesting clusters revealing concurrently changing transcript expressions across treatments were selected, characterized by a general shift of expression throughout premoult and early postmoult vs. intermoult, or by different premoult vs. postmoult expressions. A number of genes were differentially expressed in 20-hydroxyecdysone-injected crayfish vs. X-organ sinus gland extirpated males.

Diversity of LEF/TCF action in development and disease.

Lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) mediate Wnt signals in the nucleus by recruiting beta-catenin and its co-activators to Wnt response elements (WREs) of target genes. This activity is important during development but its misregulation plays a role in disease such as cancer, where overactive Wnt signaling drives LEF/TCFs to transform cells. The size of the LEF/TCF family is small: approximately four members in vertebrates and one orthologous form in flies, worms and hydra. However, size belies complexity. The LEF/TCF family exhibits extensive patterns of alternative splicing, alternative promoter usage and activities of repression, as well as activation. Recent work from numerous laboratories has highlighted how this complexity has important biological consequences in development and disease.

Biodiversity of CYP51 in trypanosomes.

Sterol 14alpha-demethylases (CYP51) are metabolic cytochromes P450, found in each biological kingdom. They catalyse a single three-step reaction included in all sterol biosynthetic pathways. Plant CYP51s have strict preference towards their physiological substrate O (obtusifoliol), which is C-4-monomethylated. Natural substrates of animal/fungal CYP51 (lanosterol, 24,25-dihydrolanosterol or 24-methylenelanosterol) are C-4-dimethylated. CYP51 from the pathogenic protozoa TB (Trypanosoma brucei) is the first example of O-specific sterol 14alpha-demethylase in non-photosynthetic organisms. Surprisingly, at 83% amino acid identity to the TB orthologue, CYP51 from TC (Trypanosoma cruzi) clearly prefers C-4-dimethylated sterols. Replacement of animal/fungi-like Ile(105) in the B' helix of TC CYP51 with phenylalanine, the residue found in this position in all plant and other trypanosome CYP51s, dramatically increases the ability of the enzyme to metabolize O, converting it into a more plant-like sterol 14alpha-demethylase. A more than 100-fold increase in binding and turnover is observed for the 24-desmethyl analogue of O [N (norlanosterol)], which is found in vivo in procyclic forms of TB and is a good TB CYP51 substrate in vitro. We believe that (i) N is a non-conventional CYP51 substrate, preferred in TB and perhaps other Trypanosomatidae and (ii) functional similarity of TC CYP51 to animal/fungal orthologues is a result of evolutionary convergence (including F105I mutation), leading to different pathways for sterol production in TC versus TB.

Understanding electron transport systems of Streptomyces cytochrome P450.

Streptomyces spp. are known to produce various types of biologically active compounds including antibiotics, antiparasitic agents, herbicides and immunosuppressants. P450 (cytochrome P450) enzymes may have key roles in these biosynthetic and biotransformation reactions. Recent genomic analysis of Streptomyces coelicolor A3(2) indicates that S. coelicolor may have six ferredoxins (Fdxs), four putative Fdx reductases (FdRs) and 18 P450 genes. However, there are few clues to explain the mechanisms and functions of Streptomyces P450 systems. To solve these questions, we have expressed and purified five S. coelicolor P450s, four FdRs and six Fdxs in Escherichia coli. Of the purified P450s, CYP105D5 has fatty acid hydroxylation activity in a system reconstituted with putidaredoxin reductase and Fdx4 or with spinach FdR and spinach Fdx, although the reconstitutions with FdR2 or FdR3 and any of the Fdxs did not support CYP105D5-catalysed oleic acid hydroxylation. Elucidation of the detailed mechanisms of electron transport system for Streptomyces P450 may provide the perspective for usefulness of P450s as a biocatalyst.

Evaluation of the PillCam Colon capsule in the detection of colonic pathology: results of the first multicenter, prospective, comparative study.

BACKGROUND AND STUDY AIMS: Population-based screening for colorectal cancer is widely recommended, with conventional colonoscopy considered to be the preferred diagnostic modality. However, compliance with screening colonoscopy is low and manpower capacity is limited. Capsule endoscopy might therefore represent a desirable alternative strategy. PATIENTS AND METHODS: The PillCam Colon capsule endoscope was prospectively tested in a multicenter setting. The indications for endoscopy in the enrolled patients included colorectal cancer screening (43 %), postpolypectomy surveillance (26 %), and lower gastrointestinal signs and symptoms (31 %). Study subjects underwent colon preparation and then ingested the capsule on the morning of the examination, with conventional colonoscopy being performed the same day. The PillCam Colon capsule findings were reviewed by three experts in capsule endoscopy who were blinded to the conventional colonoscopy findings. RESULTS: A total of 91 subjects were enrolled in three Israeli centers (55 men, 36 women; mean age 57), and the results were evaluable in 84 cases. The capsule was excreted within 10 hours in 74 % of the patients and reached the rectosigmoid colon in the other 16 %. Of the 84 evaluable patients, 20 (24 %) had significant findings, defined as at least one polyp of 6 mm or more in size or three or more polyps of any size: 14/20 (70 %) were identified with the capsule and 16/20 (80 %) were identified by conventional colonoscopy. Polyps of any size were found in 45 patients, 34/45 (76 %) found by the capsule and 36/45 (80 %) by conventional colonoscopy. In comparison with conventional colonoscopy, false-positive findings on PillCam Colon capsule examination were recorded in 15/45 cases (33 %). There were no adverse events related to the capsule endoscopy. CONCLUSIONS: PillCam Colon capsule endoscopy appears to be a promising new modality for colonic evaluation. Further improvements in the procedure will probably increase capsule examination completion and polyp detection rates. Additional studies are needed to evaluate the accuracy of PillCam Colon endoscopy in other patient populations with different prevalence levels of colonic disease.

Using behavioral science to improve fire escape behaviors in response to a smoke alarm.

Although the likelihood of fire-related death in homes with smoke alarms is about one-half that in homes without alarms, alarm effectiveness is limited by behavior. Only 16% of residents of homes with alarms have developed and practiced plans for escape when the alarm sounds. We reviewed literature to identify behavioral constructs that influence smoke alarm use. We then convened experts in the behavioral aspects of smoke alarms who reviewed the constructs and determined that the appropriate areas for behavioral focus were formulating, practicing, and implementing escape plans should an alarm sound. They subsequently identified important behaviors to be addressed by burn-prevention programs and incorporated the constructs into a behavioral model for use in such programs. Finally, we organized the available literature to support this model and make programmatic recommendations. Many gaps remain in behavioral research to improve fire escape planning and practice. Future research must select the target behavior, apply behavioral theories, and distinguish between initiation and maintenance of behaviors associated with planning, practicing, and implementing home fire escape plans.

cAMP-dependent transcription of steroidogenic genes in the human adrenal cortex requires a dual-specificity phosphatase in addition to protein kinase A.

Steroid hormone biosynthesis in the adrenal cortex is controlled by the peptide hormone adrenocorticotropin (ACTH), which acts to increase intracellular cAMP and results in the activation of cAMP-dependent protein kinase A (PKA) and subsequent increase in steroidogenic gene transcription. Protein phosphorylation by PKA activates transcription of genes encoding steroidogenic enzymes; however the precise proteins which are phosphorylated remain to be determined. We have recently shown that phosphoprotein phosphatase (PP) activity is essential for cAMP-dependent transcription of the human CYP17 (hCYP17) gene in H295R adrenocortical cells. The aim of our current studies was to determine if inhibition of PP activity attenuates cAMP-dependent mRNA expression of other steroidogenic genes in H295R cells. Using various inhibitors of serine/threonine and tyrosine PPs, we examined the role of phosphatase activity on cAMP-dependent transcription of steroidogenic genes in the adrenal cortex. CYP11A, CYP11B1/2, CYP21, and adrenodoxin also require PP activity for cAMP-stimulated gene expression. Inhibition of both serine/threonine and tyrosine PP activities suppresses the cAMP-dependent mRNA expression of several steroidogenic genes, suggesting that a dual-specificity PP is essential for conveying ACTH/cAMP-stimulated transcription. We propose that PKA phosphorylates and activates a dual-specificity phosphatase, which mediates steroidogenic gene transcription in response to ACTH/cAMP.

Expression of Wnt ligands and Frizzled receptors in colonic mucosa and in colon carcinoma.

AIMS: Signalling through the Wnt pathway is integrally associated with colon carcinogenesis. Although activating mutations in the genes for adenomatous polyposis coli (APC) and beta-catenin are clearly associated with colon cancer, less is understood about the role of the upstream secreted ligands (Wnts) and their receptors (frizzled, Fz) in this process. In other systems, increased Wnt signalling has been shown to alter the expression of components of this pathway. This study was designed to test the hypothesis that colon cancer is characterised by aberrant expression of specific Wnt genes and Fz receptors. METHODS: The expression of Wnt genes was assessed by in situ, antisense RNA hybridisation in paraffin wax embedded samples of normal and malignant human colon tissues with probes specific for the individual Wnt genes. The expression of Fz1 and Fz2 was determined by immunoperoxidase based antibody staining on human tissues. RESULTS: Changes in the expression of some ligands and receptors were seen in colon cancer. For example, Wnt2 mRNA was detected in colon cancer but was undetectable in normal colonic mucosa. Differential expression of Wnt5a in normal mucosa was also noted, with increased expression at the base of the crypts compared with the luminal villi and slightly increased expression in colon cancer. Wnt7a exhibited minimal expression in both normal and malignant colon tissues, whereas other Wnt ligands including Wnts 1, 4, 5b, 6, 7b, and 10b were expressed equally and strongly in both normal and malignant colon tissues. In defining cellular responses and phenotype, the type and distribution of Fz receptors may be as important as the pattern of Wnt ligand expression. No expression of Fz receptor 1 and 2 was seen in normal colonic mucosa and in well differentiated tumours. However, poorly differentiated tumours exhibited a high degree of Fz receptor expression, especially at the margin of cellular invasion. CONCLUSIONS: These data indicate that the expression of members of the Wnt signal transduction pathway, distinct from APC and beta-catenin, is integrally associated with the process of colon carcinogenesis. Wnt2, and possibly Wnt5a, may be involved in the progression from normal mucosa to cancer and the expression of Fz1/2 receptors may be involved in processes associated with tumour invasion. Altered expression of these Wnts and Fz receptors may prove useful as prognostic or diagnostic markers for patients with colon cancer.