The study was designed to determine the effects of peripheral injection of SB203580 on the synthesis of interleukin- (IL-) 1β, IL-6, and tumor necrosis factor (TNF) α in the hypothalamus of ewes during prolonged inflammation. Inflammation was induced by the administration of lipopolysaccharide (LPS) (400 ng/kg) over 7 days. SB203580 is a selective ATP-competitive inhibitor of the p38 mitogen-activated protein kinase (MAPK), which is involved in the regulation of proinflammatory cytokines IL-1β, IL-6 and TNFα synthesis. Intravenous injection of SB203580 successfully inhibited (P < 0.01) synthesis of IL-1β and reduced (P < 0.01) the production of IL-6 in the hypothalamus. The p38 MAPK inhibitor decreased (P < 0.01) gene expression of TNFα but its effect was not observed at the level of TNFα protein synthesis. SB203580 also reduced (P < 0.01) LPS-stimulated IL-1 receptor type 1 gene expression. The conclusion that inhibition of p38 MAPK blocks LPS-induced proinflammatory cytokine synthesis seems to initiate new perspectives in the treatment of chronic inflammatory diseases also within the central nervous system. However, potential proinflammatory effects of SB203580 treatment suggest that all therapies using p38 MAPK inhibitors should be introduced very carefully with analysis of all expected and unexpected consequences of treatment.
The cosmetic industry adapts to the needs of consumers seeking to limit the use of preservatives and develop of preservative-free or self-preserving cosmetics, where preservatives are replaced by raw materials of plant origin. The aim of study was a comparison of the antimicrobial activity of extracts (Matricaria chamomilla, Aloe vera, Calendula officinalis) and essential oils (Lavandulla officinallis,Melaleuca alternifolia, Cinnamomum zeylanicum) with methylparaben. Extracts (2.5 %), essential oils (2.5 %) and methylparaben (0.4 %) were tested against Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC25922,Staphylococcus aureus ATCC 29213, Candida albicans ATCC 14053. Essentials oils showed higher inhibitory activity against tested microorganism strain than extracts and methylparaben. Depending on tested microorganism strain, all tested extracts and essential oils show antimicrobial activity 0.8–1.7 and 1–3.5 times stronger than methylparaben, respectively. This shows that tested extracts and essential oils could replace use of methylparaben, at the same time giving a guarantee of microbiological purity of the cosmetic under its use and storage.
Antimicrobial activity; Essential oils; Herbal extracts; Methylparaben
This study was performed to evaluate dosimetric differences between current intensity modulated radiation therapy (IMRT) delivery modes: Step-and-shoot (SS), sliding window (SW), and volumetric modulated arc therapy (VMAT). Plans for 15 prostate cancer patients with 10 MV photon beams using each IMRT mode were generated. Patients had three planning target volumes (PTVs) including prostate, prostate plus seminal vesicles, and pelvic lymphatics. Dose volume histograms (DVHs) of PTVs and organs at risk (OARs), tumor control probability (TCP) and normal tissue complication probabilities (NTCPs), conformation number, and monitor units (MUs) used were compared. Statistical analysis was performed using the analysis of variance (ANOVA) technique. The TCPs were < 99% with insignificant differences among modalities (P > 0.99). Doses to all critical structures were higher on average with SW method compared to SS, but insignificant. NTCP values were lowest for VMAT in all structures excepting bladder. Normal tissue volumes receiving doses in the 20-30 Gy range were reduced for VMAT compared to SS. Percentage of MUs required for VMAT to deliver a comparable plan to SS and SW was at least 40% less. In conclusion, similar target coverage and normal tissue doses were found by the three compared modes and the dosimetric differences were small.
Intensity modulated radiation therapy; prostate cancer; step-and-shoot; sliding window; volumetric modulated arc therapy
Acting at the level of the brain, interleukin- (IL-)1β is considered to be one of the most potent downregulators of reproduction processes during immune/inflammatory challenge. IL-1β suppresses gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus resulting in the inhibition of the luteinizing hormone (LH) release from the anterior pituitary (AP). However, the presence of IL-1β receptors in the AP suggests the possible direct action of this cytokine on LH secretion. The study was designed to determine the effect of IL-1β on the LH secretion from the AP explants collected from saline and LPS-treated ewes in the follicular phase. It was found that IL-1β suppressed (P ≤ 0.01) GnRH-stimulated LH release and LHβ gene expression in AP explants in both groups. However, IL-1β action was more potent in the explants collected from LPS-treated animals. Pituitaries from LPS-treated animals were characterized by increased (P ≤ 0.01) IL-1 type I receptor and decreased (P ≤ 0.01) GnRH receptor gene expression level compared to the saline-treated group. IL-1β also affected the GnRH-R gene expression in explants collected from LPS-treated animals. Our results show that direct action of IL-1β on the pituitary gonadotropes could be one of the reasons of the reproductive processes disorders accompanying an inflammatory state.
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
LC3; autolysosome; autophagosome; flux; lysosome; phagophore; stress; vacuole
The aim of this study was to evaluate differences in dose distributions in stereotactic body radiation therapy treatment plans for lung tumors calculated with pencil beam convolution (PBC) algorithm with modified Batho power law (MBPL) versus heterogeneity corrected anisotropic analytical algorithm (AAA) of the Varian Eclipse treatment planning system. The four-dimensional computed tomography images from 20 patients with lung cancer were used to create treatment plans. Plans used five to seven nonopposing coplanar 6 MV beams. Plans generated with the PBC algorithm and MBPL for tissue heterogeneity corrections were optimized to deliver 60 Gy in three fractions to at least 95% of the planned target volume, and the normal tissue doses for spinal cord, esophagus, heart, and ipsilateral bronchus were restricted to less than 18, 27, 30, and 30 Gy, respectively. Plans were recalculated with AAA, retaining identical beam arrangements, photon beam fluences, and monitor units. The pencil beam plans, designed to deliver 60 Gy, delivered on average 51.6 Gy when re-calculated with the AAA, suggesting a reduction of at least 10% to prescription dose is appropriate when calculating with the AAA.
Anisotropic analytical algorithm; dose calculation algorithm; lung cancer; pencil beam convolution
This study aims at evaluating the impact of tissue heterogeneity corrections on dosimetry of stereotactic body radiation therapy treatment plans. Four-dimensional computed tomography data from 15 low stage non-small cell lung cancer patients was used. Treatment planning and dose calculations were done using pencil beam convolution algorithm of Varian Eclipse system with Modified Batho Power Law for tissue heterogeneity. Patient plans were generated with 6 MV co-planar non-opposing four to six field beams optimized with tissue heterogeneity corrections to deliver a prescribed dose of 60 Gy in three fractions to at least 95% of the planning target volume, keeping spinal cord dose <10 Gy. The same plans were then regenerated without heterogeneity correction by recalculating previously optimized treatment plans keeping identical beam arrangements, field fluences and monitor units. Compared with heterogeneity corrected plans, the non-corrected plans had lower average minimum, mean, and maximum tumor doses by 13%, 8%, and 6% respectively. The results indicate that tissue heterogeneity is an important determinant of dosimetric optimization of SBRT plans.
Heterogeneity corrections; NSCLC; SBRT
The effect of oral folic acid on jejunal glycolytic enzyme activity in five fasting obese patients and in three normal male volunteers on a constant 3000 cal diet was studied. The glycolytic enzymes, fructokinase, hexokinase, glucokinase, fructose-1-phosphate aldolase, and fructose diphosphate aldolase, and the disaccharidases, sucrase, maltase, and lactase were measured.
In both the fasting patients and the normal volunteers, oral folic acid significantly increased the jejunal glycolytic enzyme activities but had no effect on disaccharidase activity. When oral folic acid was discontinued in the normal volunteers, the glycolytic enzyme activities returned to control values. In the obese patients, refeeding and folic acid caused a further increase in glycolytic enzyme activities above that seen with fasting and folic acid.
In contrast to oral folic acid, intramuscular folic acid, oral vitamin B12, and oral tetracycline had no effect on glycolytic enzyme activities.
These studies demonstrate that oral folic acid which is neither a substrate nor a coenzyme of these enzymes, increases human jejunal glycolytic enzyme activity in a specific fashion. This would appear to be an action of oral folic acid which has not been recognized previously.
Marine protected areas (MPAs) are key tools for combatting the global overexploitation of endangered species. The prevailing paradigm is that MPAs are beneficial in helping to restore ecosystems to more ‘natural’ conditions. However, MPAs may have unintended negative effects when increasing densities of protected species exert destructive effects on their habitat. Here, we report on severe seagrass degradation in a decade-old MPA where hyper-abundant green turtles adopted a previously undescribed below-ground foraging strategy. By digging for and consuming rhizomes and roots, turtles create abundant bare gaps, thereby enhancing erosion and reducing seagrass regrowth. A fully parametrized model reveals that the ecosystem is approaching a tipping point, where consumption overwhelms regrowth, which could potentially lead to complete collapse of the seagrass habitat. Seagrass recovery will not ensue unless turtle density is reduced to nearly zero, eliminating the MPA's value as a turtle reserve. Our results reveal an unrecognized, yet imminent threat to MPAs, as sea turtle densities are increasing at major nesting sites and the decline of seagrass habitat forces turtles to concentrate on the remaining meadows inside reserves. This emphasizes the need for policy and management approaches that consider the interactions of protected species with their habitat.
marine reserves; plant–herbivore interactions; alternate stable states
The virulence of Salmonella is linked to its invasive capacity and suppression of adaptive immunity. This does not explain, however, the rapid dissemination of the pathogen after breaching the gut. Here we showed that early in infection, S. Typhimurium suppressed degranulation of local mast cells (MCs), resulting in limited neutrophil recruitment and restricted outflow of vascular contents into infection sites, thus facilitating bacterial spread. MC suppression was mediated by the Salmonella phosphatase (SptP), which shares structural homology with Yersinia YopH. SptP functioned by dephosphorylating the vesicle fusion protein N-ethylmalemide-sensitive factor (NSF) and by blocking phosphorylation of Syk. Without SptP, orally challenged S. Typhimurium failed to suppress MC degranulation and exhibited limited colonization of the mesenteric lymph nodes. Administration of SptP to sites of Escherichia coli infection markedly enhanced its virulence. Thus, SptP-mediated inactivation of local MCs is a powerful mechanism utilized by S. Typhimurium to impede early innate immunity.
To determine if processes and outcomes of diabetes care improved between 2000 and 2006 in a managed care health plan with a comprehensive diabetes disease management program.
A total of 1650 randomly selected members with diabetes mellitus completed surveys in 2000, and 1256 randomly selected members with diabetes completed surveys in 2006. Survey and medical record data were analyzed using multivariable regression and predictive probabilities adjusted for age, education, and comorbidities.
In 2006, patients were more likely to have proteinuria assessed (85% vs 74% in 2000), foot examinations performed (90% vs 86%), glycosylated hemoglobin levels measured (94% vs 87%), lipids measured (81% vs 70%), aspirin use recommended (67% vs 56%), and influenza immunizations administered (70% vs 63%). Glycosylated hemoglobin levels decreased by 0.60% (P <.001), systolic blood pressures by 3 mm Hg (P = .002), and low-density lipoprotein cholesterol levels by 18 mg/dL (P <.001). Those who were continuously enrolled in the health plan were significantly more likely to report having had dilated retinal examinations (P = .003), aspirin use recommendations (P = .049), influenza immunizations (P = .004), and lower low-density lipoprotein cholesterol levels (by 6 mg/dL, P = .003).
Implementation of a disease management program was associated with substantial improvements in processes and outcomes of diabetes care over 6 years. Although secular trend likely contributed somewhat, improvement in other measures was significantly associated with duration of enrollment in the health plan, making secular trend an unlikely explanation for all of our findings.
During mitosis, the spindle assembly checkpoint (SAC) monitors the attachment of kinetochores (KTs) to the plus ends of spindle microtubules (MTs) and prevents anaphase onset until chromosomes are aligned and KTs are under proper tension. Here, we identify a SAC component, BuGZ/ZNF207, from an RNAi viability screen in human Glioblastoma multiforme (GBM) brain tumor stem cells. BuGZ binds to and stabilizes Bub3 during interphase and mitosis through a highly conserved GLE2p-binding sequence (GLEBS) domain. Inhibition of BuGZ results in loss of both Bub3 and its binding partner Bub1 from KTs, reduction of Bub1-dependent phosphorylation of centromeric histone H2A, attenuation of KT-based Aurora kinase B activity, and lethal chromosome congression defects in cancer cells. Phylogenetic analysis indicates that BuGZ orthologs are highly conserved among eukaryotes, but are conspicuously absent from budding and fission yeasts. These findings suggest BuGZ has evolved to facilitate Bub3 activity and chromosome congression in higher eukaryotes.
BuGZ; ZNF207; Bub3; spindle assembly checkpoint; kinetochore; cancer stem cells; Glioblastoma multiforme
Long-term survival rates for patients with resected pancreatic ductal adenocarcinoma (PDAC) have stagnated at 20% for more than a decade, demonstrating the need to develop novel adjuvant therapies. Gemcitabine-erlotinib therapy has demonstrated a survival benefit for patients with metastatic PDAC. Here we report the first phase 2 study of erlotinib in combination with adjuvant chemoradiation and chemotherapy for resected PDAC.
Methods and Materials
Forty-eight patients with resected PDAC received adjuvant erlotinib (100 mg daily) and capecitabine (800 mg/m2 twice daily Monday-Friday) concurrently with intensity modulated radiation therapy (IMRT), 50.4 Gy over 28 fractions followed by 4 cycles of gemcitabine (1000 mg/m2 on days 1, 8, and 15 every 28 days) and erlotinib (100 mg daily). The primary endpoint was recurrence-free survival (RFS).
The median follow-up time was 18.2 months (interquartile range, 13.8–27.1). Lymph nodes were positive in 85% of patients, and margins were positive in 17%. The median RFS was 15.6 months (95% confidence interval [CI], 13.4–17.9), and the median overall survival (OS) was 24.4 months (95% CI, 18.9–29.7). Multivariate analysis with adjustment for known prognostic factors showed that tumor diameter >3 cm was predictive for inferior RFS (hazard ratio, 4.01; P = .001) and OS (HR, 4.98; P = .02), and the development of dermatitis was associated with improved RFS (HR, 0.27; P = .009). During CRT and post-CRT chemotherapy, the rates of grade 3/4 toxicity were 31%/2% and 35%/8%, respectively.
Erlotinib can be safely administered with adjuvant IMRT-based CRT and chemotherapy. The efficacy of this regimen appears comparable to that of existing adjuvant regimens. Radiation Therapy Oncology Group 0848 will ultimately determine whether erlotinib produces a survival benefit in patients with resected pancreatic cancer.
In The Netherlands, standard prehospital trauma care is provided by emergency medical services and can be supplemented with advanced trauma care by Mobile Medical Teams. Due to observed over and undertriage in the dispatch of the Mobile Medical Team for major trauma patients, the accuracy of the dispatch criteria has been disputed. In order to obtain recommendations to invigorate the dispatch criteria, this study aimed at reaching consensus in expert opinion on the question; which acute trauma patient is in need of care by a Mobile Medical Team? In this paper we describe the protocol of the DENIM study (a Delphi-procedure on the identification of prehospital trauma patients in need of care by Mobile Medical Teams).
A national three round digital Delphi study will be conducted to reach consensus. Literature was explored for relevant topics. After agreement on the themes of interest, the steering committee will construct questions for the first round. In total, 120 panellists with the following backgrounds; Mobile Medical Team physicians and nurses, trauma surgeons, ambulance nurses, emergency medical operators will be invited to participate. Group opinion will be fed back between each round that follows, allowing the panellists to revise their previous opinions and so, converge towards group consensus.
Successful prehospital treatment of trauma patients greatly depends on the autonomous decisions made by the different professionals along the chain of prehospital trauma care. Trauma patients in need of care by the Mobile Medical Team need to be identified by those professionals in order to invigorate deployment criteria and improve trauma care. The Delphi technique is used because it allows for group consensus to be reached in a systematic and anonymous fashion amongst experts in the field of trauma care. The anonymous nature of the Delphi allows all experts to state their opinion whilst eliminating the bias of dominant and/or hierarchical individuals on group opinion.
DENIM; Delphi; Protocol; Dispatch; Trauma; Mobile Medical Team; Helicopter; Ambulance
We report the presence of West Nile virus in a cryopreserved, dengue-negative serum specimen collected from an acute fever case on Java in 2004–2005. The strain belongs to genotype lineage 2, which has recently been implicated in human outbreaks in Europe.
Identifying biomarkers in body fluids may improve the noninvasive detection of colorectal cancer. Previously, we identified N-Myc downstream-regulated gene 4 (NDRG4) and GATA binding protein 5 (GATA5) methylation as promising biomarkers for colorectal cancer in stool DNA. Here, we examined the utility of NDRG4, GATA5, and two additional markers [Forkhead box protein E1 (FOXE1) and spectrin repeat containing nuclear envelope 1 (SYNE1)] promoter methylation as biomarkers in plasma DNA. Quantitative methylation-specific PCR was performed on plasma DNA from 220 patients with colorectal cancer and 684 noncancer controls, divided in a training set and a test set. Receiver operating characteristic analysis was performed to measure the area under the curve of GATA5, NDRG4, SYNE1, and FOXE1 methylation. Functional assays were performed in SYNE1 and FOXE1 stably transfected cell lines. The sensitivity of NDRG4, GATA5, FOXE1, and SYNE1 methylation in all stages of colorectal cancer (154 cases, 444 controls) was 27% [95% confidence interval (CI), 20%–34%), 18% (95% CI, 12%–24%), 46% (95% CI, 38%– 54%), and 47% (95% CI, 39%–55%), with a specificity of 95% (95% CI, 93%–97%), 99% (95% CI, 98%–100%), 93% (95% CI, 91%–95%), and 96% (95% CI, 94%–98%), respectively. Combining SYNE1 and FOXE1, increased the sensitivity to 56% (95% CI, 48%–64%), while the specificity decreased to 90% (95% CI, 87%–93%) in the training set and to 58% sensitivity (95% CI, 46%–70%) and 91% specificity (95% CI, 80%–100%) in a test set (66 cases, 240 controls). SYNE1 overexpression showed no major differences in cell proliferation, migration, and invasion compared with controls. Overexpression of FOXE1 significantly decreased the number of colonies in SW480 and HCT116 cell lines. Overall, our data suggest that SYNE1 and FOXE1 are promising markers for colorectal cancer detection.
Nineteen species of abundant gastropods were collected at Robben Island, including introduced dune snails and European brown garden snails. They were identified using morphology and DNA barcoding. It was expected that the species recorded would be similar to those from the Cape peninsula, South Africa, but we were surprised to find some exceptions: the very abundant invasive mussel species in South Africa, the South American bisexual mussel (Semimytilus
algosus), and the beaded topshells (Oxystele
impervia) were not found on Robben Island. Possible explanations are presented for these differences.
Mollusca; Gastropoda; mitochondrial gene COI; species identification
Brain dopamine (DA) plays a central role in addictive disorders including nicotine addiction. Several DA-related gene variants have been studied to identify predictors of pharmacotherapy response for smoking cessation. Genetic variants in DRD2, DRD4, ANKK1, DAT1, COMT and DBH genes show some promise in predicting response to smoking cessation pharmacotherapies. However, most of the clinical trials studying these variants had small sample sizes, used retrospective analysis, and enrolled smokers of self-identified Caucasians as study participants. In addition, future studies should also consider nicotine metabolism rate, sex or menstrual cycle phase, and epigenetic factors as potential confounding factors for treatment response of smokers. Future work addressing these limitations may uncover the potential impact of DA genetic variation on smoking cessation pharmacotherapies.
Pharmacogenetics; Nicotine; Dopamine; Smoking Cessation; Genetic; Addiction
Genome sequencing can provide rapid insights on transmission and pathogenesis of emerging pathogens.
Elizabethkingia anophelis, recently discovered from mosquito gut, is an emerging bacterium associated with neonatal meningitis and nosocomial outbreaks. However, its transmission route remains unknown. We use rapid genome sequencing to investigate 3 cases of E. anophelis sepsis involving 2 neonates who had meningitis and 1 neonate’s mother who had chorioamnionitis. Comparative genomics revealed evidence for perinatal vertical transmission from a mother to her neonate; the 2 isolates from these patients, HKU37 and HKU38, shared essentially identical genome sequences. In contrast, the strain from another neonate (HKU36) was genetically divergent, showing only 78.6% genome sequence identity to HKU37 and HKU38, thus excluding a clonal outbreak. Comparison to genomes from mosquito strains revealed potential metabolic adaptations in E. anophelis under different environments. Maternal infection, not mosquitoes, is most likely the source of neonatal E. anophelis infections. Our findings highlight the power of genome sequencing in gaining rapid insights on transmission and pathogenesis of emerging pathogens.
Elizabethkingia anophelis; neonatal; meningitis; sepsis; vertical; transmission; maternal; bacteria; genome; mother; child; Hong Kong; sequencing
To determine the prevalence and sensitivity of CT Colonography (CTC) in the detection of non-polypoid adenomas using restricted criteria of height:width ratio of <50% and height elevation of ≤3mm.
MATERIAL AND METHODS
In ACRIN 6664, an institutional review board-approved, HIPAA-compliant study, a cohort of 2531 asymptomatic participants underwent CTC and screening colonoscopy. The CTC exams were interpreted by both two-dimensional and three-dimensional techniques. Non-polypoid adenomatous polyps identified by CTC or colonoscopy were retrospectively reviewed to determine which polyps met the restricted criteria. The prevalence of non-polypoid adenomas and prospective sensitivity of CTC were determined. Descriptive statistics are used to report the prevalence, size, and histology. Sensitivities for the non-polypoid (with 95% CIs) and polypoid lesions are compared with a two-sided Z test for two independent binomial proportions.
The retrospective review confirmed 21 non-polypoid adenomas, yielding a prevalence of 0.83% (21/2531 participants). 8 (38.1%) were advanced adenomas, many (50%, 4/8) secondary to large size (≥10mm) only. The overall per polyp sensitivity of CTC (combined 2D and 3D interpretation) for detecting non-polypoid adenomas ≥ 5mm (n=21), ≥ 6mm (n=16) and ≥10mm (n=5) were 0.76, 0.75, and 0.80, respectively, which was not statistically different from the sensitivity of detecting polypoid adenomas (p>0.37).
In this large screening population, non-polypoid adenomas had a very low prevalence (<1%), and advanced pathologic features were uncommon in polyps <10mm in diameter. The majority of non-polypoid adenomas are technically visible at CTC, with prospective sensitivities similar to polypoid adenomas using an interpretation approach combining both two-dimensional and three-dimensional review.
Identifying the mechanisms by which cells remain irreversibly committed to their fates is a critical step toward understanding and being able to manipulate development and homeostasis. Polycomb group (PcG) proteins are chromatin modifiers that maintain transcriptional silencing, and loss of PcG genes causes widespread derepression of many developmentally important genes. However, because of their broad effects, the degree to which PcG proteins are used at specific fate choice points has not been tested. To understand how fate choices are maintained, we have been analyzing R7 photoreceptor neuron development in the fly eye. R1, R6, and R7 neurons are recruited from a pool of equivalent precursors. In order to adopt the R7 fate, these precursors make three binary choices. They: (1) adopt a neuronal fate, as a consequence of high receptor tyrosine kinase (RTK) activity (they would otherwise become non-neuronal support cells); (2) fail to express Seven-up (Svp), as a consequence of Notch (N) activation (they would otherwise express Svp and become R1/R6 neurons); and (3) fail to express Senseless (Sens), as a parallel consequence of N activation (they would otherwise express Sens and become R8 neurons in the absence of Svp). We were able to remove PcG genes specifically from post-mitotic R1/R6/R7 precursors, allowing us to probe these genes' roles in the three binary fate choices that R1/R6/R7 precursors face when differentiating as R7s.
Here, we show that loss of the PcG genes Sce, Scm, or Pc specifically affects one of the three binary fate choices that R7 precursors must make: mutant R7s derepress Sens and adopt R8 fate characteristics. We find that this fate transformation occurs independently of the PcG genes' canonical role in repressing Hox genes. While N initially establishes Sens repression in R7s, we show that N is not required to keep Sens off, nor do these PcG genes act downstream of N. Instead, the PcG genes act independently of N to maintain Sens repression in R1/R6/R7 precursors that adopt the R7 fate.
We conclude that cells can use PcG genes specifically to maintain a subset of their binary fate choices.
Polycomb; PRC1; Photoreceptor; R7; Fate switch; Stochastic; Notch; Senseless/Gfi-1
Over the last decade genetic marker-based plant breeding strategies have gained increasing attention because genotyping technologies are no longer limiting. Now the challenge is to optimally use genetic markers in practical breeding schemes. For simple traits such as some disease resistances it is possible to target a fixed multi-locus allele configuration at a small number of causal or linked loci. Efficiently obtaining this genetic ideotype from a given set of parental genotypes is known as the marker-assisted gene pyramiding problem. Previous methods either imposed strong restrictions or used black box integer programming solutions, while this paper explores the power of an explicit heuristic approach that exploits the underlying genetic structure to prune the search space.
Gene Stacker is introduced as a novel approach to marker-assisted gene pyramiding, combining an explicit directed acyclic graph model with a pruned generation algorithm inspired by a simple exhaustive search. Both exact and heuristic pruning criteria are applied to reduce the number of generated schedules. It is shown that this approach can effectively be used to obtain good solutions for stacking problems of varying complexity. For more complex problems, the heuristics allow to obtain valuable approximations. For smaller problems, fewer heuristics can be applied, resulting in an interesting quality-runtime tradeoff. Gene Stacker is competitive with previous methods and often finds better and/or additional solutions within reasonable time, because of the powerful heuristics.
The proposed approach was confirmed to be feasible in combination with heuristics to cope with realistic, complex stacking problems. The inherent flexibility of this approach allows to easily address important breeding constraints so that the obtained schedules can be widely used in practice without major modifications. In addition, the ideas applied for Gene Stacker can be incorporated in and extended for a plant breeding context that e.g. also addresses complex quantitative traits or conservation of genetic background. Gene Stacker is freely available as open source software at http://genestacker.ugent.be. The website also provides documentation and examples of how to use Gene Stacker.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-014-0154-z) contains supplementary material, which is available to authorized users.
Plant breeding; Marker-assisted gene pyramiding; Multi-objective optimization; Heuristics
Pancreatic cancer is the fourth leading cause of cancer deaths and there currently is no reliable modality for the early detection of this disease. Here we identify cancer-specific promoter DNA methylation of BNC1 and ADAMTS1 as a promising biomarker detection strategy meriting investigation in pancreatic cancer.
We used a genome-wide pharmacologic transcriptome approach to identify novel cancer-specific DNA methylation alterations in pancreatic cancer cell lines. Of 8 promising genes, we focused our studies on BNC1 and ADAMTS1 for further downstream analysis including methylation and expression. We used a nanoparticle-enabled MOB (Methylation On Beads) technology to detect early stage pancreatic cancers by analyzing DNA methylation in patient serum.
We identified 2 novel genes, BNC1 (92%) and ADAMTS1, (68%) that showed a high frequency of methylation in pancreas cancers (n=143), up to 100% in PanIN-3 and 97% in Stage I invasive cancers. Using the nanoparticle-enabled MOB technology, these alterations could be detected in serum samples (n=42) from pancreas cancer patients, with a sensitivity for BNC1 of 79% (95%CI:66-91%) and for ADAMTS1 of 48% (95%CI:33-63%), while specificity was 89% for BNC1 (95%CI:76-100%) and 92% for ADAMTS1 (95%CI:82-100%). Overall sensitivity using both markers is 81% (95%CI:69-93%) and specificity is 85% (95%CI:71-99%).
Promoter DNA methylation of BNC1 and ADAMTS1 are potential biomarkers to detect early stage pancreatic cancers. Assaying the promoter methylation status of these genes in circulating DNA from serum is a promising strategy for early detection of pancreatic cancer and has the potential to improve mortality from this disease.
DNA Methylation; Early detection biomarker; Pancreatic cancer; Cancer screening
The prevalence of the metabolic syndrome and underlying metabolic disturbances increase rapidly in developed countries. Various molecular targets are currently under investigation to unravel the molecular mechanisms that cause these disturbances. This is done in attempt to counter or prevent the negative health consequences of the metabolic disturbances. Here, we reviewed the current knowledge on the role of C/EBP-β in these metabolic disturbances. C/EBP-β deletion in mice resulted in downregulation of hepatic lipogenic genes and increased expression of β-oxidation genes in brown adipose tissue. Furthermore, C/EBP-β is important in the differentiation and maturation of adipocytes and is increased during ER stress and proinflammatory conditions. So far, studies were only conducted in animals and in cell systems. The results found that C/EBP-β is an important transcription factor within the metabolic disturbances of the metabolic system. Therefore, it is interesting to examine the potential role of C/EBP-β at molecular and physiological level in humans.
Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β1. As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β1 induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β1. The failure by TGF-β1 to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β1 signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.
phenotype plasticity; asthma; airway remodelling; caveolae; TGF-β1