Combination of multifunctional ursolic acid with kinase inhibitors for anti-cancer drug carrier vesicles.

A sterically stabilized unilamellar nanocarrier vesicle (SSV) system containing dipalmitoylphosphatidylcholine, cholesterol, ursolic acid and PEGylated phospholipid has been developed by exploiting the structural advantages of ursolic acid: by spontaneously attaching to the lipid head groups, it induces curvature at the outer side of the bilayers, allowing the preparation of size-limited vesicles without extrusion. Ursolic acid (UA) also interacts with the PEG chains, supporting steric stabilization even when the amount of PEGylated phospholipid is reduced. Using fluorescence immunohistochemistry, vesicles containing ursolic acid (UA-SSVs) were found to accumulate in the tumor in 3 h on xenografted mouse, suggesting the potential use of these vesicles for passive tumor targeting. Further on, mono- and combination therapy with UA and six different kinase inhibitors (crizotinib, erlotinib, foretinib, gefitinib, refametinib, trametinib) was tested on seven cancer cell-lines. In most combinations synergism was observed, in the case of trametinib even at very low concentration (0.001 µM), which targets the MAPK pathway most often activated in human cancers. The coupled intercalation of UA and trametinib (2:1 molar ratio) into vesicles causes further structural advantageous molecular interactions, promoting the formation of small vesicles. The high drug:lipid molar ratio (~0.5) in the novel type of co-delivery vesicles enables their direct medical application, possibly also overcoming the multidrug resistance effect.

The Influence of Meteorological Factors and Atmospheric Pollutants on the Risk of Preterm Birth.

Atmospheric pollutants and meteorological conditions are suspected to be causes of preterm birth. We aimed to characterize their possible association with the risk of preterm birth (defined as birth occurring before 37 completed gestational weeks). We pooled individual data from 13 birth cohorts in 11 European countries (71,493 births from the period 1994-2011, European Study of Cohorts for Air Pollution Effects (ESCAPE)). City-specific meteorological data from routine monitors were averaged over time windows spanning from 1 week to the whole pregnancy. Atmospheric pollution measurements (nitrogen oxides and particulate matter) were combined with data from permanent monitors and land-use data into seasonally adjusted land-use regression models. Preterm birth risks associated with air pollution and meteorological factors were estimated using adjusted discrete-time Cox models. The frequency of preterm birth was 5.0%. Preterm birth risk tended to increase with first-trimester average atmospheric pressure (odds ratio per 5-mbar increase = 1.06, 95% confidence interval: 1.01, 1.11), which could not be distinguished from altitude. There was also some evidence of an increase in preterm birth risk with first-trimester average temperature in the -5°C to 15°C range, with a plateau afterwards (spline coding, P = 0.08). No evidence of adverse association with atmospheric pollutants was observed. Our study lends support for an increase in preterm birth risk with atmospheric pressure.

Decreased peroxisome proliferator-activated receptor γ level and signalling in sebaceous glands of patients with acne vulgaris.

Little is known about the altered lipid metabolism-related transcriptional events occuring in sebaceous glands of patients with acne vulgaris. Peroxisome proliferator-activated receptor (PPAR)γ, a lipid-activated transcription factor, is implicated in differentiation and lipid metabolism of sebocytes. We have observed that PPARγ and its target genes, ADRP (adipose differentiation related protein) and PGAR (PPARγ angioprotein related protein) are expressed at lower levels in sebocytes from patients with acne than in those from healthy controls (HCs) Furthermore, endogenous PPARγ activator lipids such as arachidonic acid-derived keto-metabolites (e.g. 5KETE, 12KETE) are increased in acne-involved and nonacne-involved skin of patients with acne, compared with skin from healthy individuals. Our findings highlight the possible anti-inflammatory role of endogenous ligand-activated PPARγ signaling in human sebocyte biology, and suggest that modulating PPARγ- expression and thereby signaling might be a promising strategy for the clinical management of acne vulgaris.

Silica@zirconia@poly(malic acid) nanoparticles: promising nanocarriers for theranostic applications.

Silica@zirconia@poly(malic acid) nanocarriers of 110 nm mean diameter were designed, synthesized and characterized for the targeted delivery of diagnostic and therapeutic 99mTc to folate-overexpressing tumors. An important achievement was that a multifunctional l-(-)-malic-acid-based copolymer was formed in situ at the surface of the inorganic cores in a single synthetic step incorporating l-(-)-malic acid, ß-cyclodextrin rings, folic acid moieties, and polyethylene glycol chains. Morphological and in-depth structural analysis of the particles proved their core@shell structure. Stability experiments in aqueous media evidenced that stable suspensions can be obtained from the lyophilized powder in 10 mM phosphate buffer at pH 7.4. During 14-day degradation experiments, the nanoparticles were found to be slowly dissolving (including inorganic core) in saline and also in total cell medium. An in vitro toxicity assay on hepatocytes showed a concentration-dependent decrease of cell viability down to 63 ± 1% at the highest applied concentration (0.5 mg ml-1). Proof of concept experiments of technetium-99m radiolabelling and in vivo labelling stability are presented.

Inherently fluorescent and porous zirconia colloids: preparation, characterization and drug adsorption studies.

Porous, fluorescent zirconia particles of nearly 380 nm diameter were prepared without template molecules or labeling dyes. The porous structure is the result of aggregation-induced particle formation. The inherent fluorescence is assigned to coordinatively unsaturated Zr4+ ions at the sol-gel derived ZrO2 surface. After physico-chemical characterization of the native zirconia particles carboxyl and/or amine bearing drug molecules (d,l-α-difluoromethylornithine - DFMO, ursolic acid - UA and doxorubicin - DOX) were adsorbed onto their surface, and the products were analyzed with Fourier-transform infrared spectroscopy (FTIR), thermogravimetry (TG), small-angle X-ray scattering (SAXS), fluorimetry and zeta potential vs. pH measurements. We have found that DOX complexes coordinatively unsaturated Zr4+ ions without dislocating them, while carboxyl-bearing drugs interact with basic surface Zr-OH sites eliminating some of the carbonate species. The adsorption of UA at the zirconia surface shifts considerably the isoelectric point of the surface and thus provides kinetic stability to the particles at physiological pH. An in vivo biodistribution study in two healthy dogs performed by SPECT/CT detection after 99mTc labeling of the nanocarriers has shown the possibility of drug delivery application.

Performance of multi-city land use regression models for nitrogen dioxide and fine particles.

BACKGROUND: Land use regression (LUR) models have been developed mostly to explain intraurban variations in air pollution based on often small local monitoring campaigns. Transferability of LUR models from city to city has been investigated, but little is known about the performance of models based on large numbers of monitoring sites covering a large area. OBJECTIVES: We aimed to develop European and regional LUR models and to examine their transferability to areas not used for model development. METHODS: We evaluated LUR models for nitrogen dioxide (NO2) and particulate matter (PM; PM2.5, PM2.5 absorbance) by combining standardized measurement data from 17 (PM) and 23 (NO2) ESCAPE (European Study of Cohorts for Air Pollution Effects) study areas across 14 European countries for PM and NO2. Models were evaluated with cross-validation (CV) and hold-out validation (HV). We investigated the transferability of the models by successively excluding each study area from model building. RESULTS: The European model explained 56% of the concentration variability across all sites for NO2, 86% for PM2.5, and 70% for PM2.5 absorbance. The HV R2s were only slightly lower than the model R2 (NO2, 54%; PM2.5, 80%; PM2.5 absorbance, 70%). The European NO2, PM2.5, and PM2.5 absorbance models explained a median of 59%, 48%, and 70% of within-area variability in individual areas. The transferred models predicted a modest-to-large fraction of variability in areas that were excluded from model building (median R2: NO2, 59%; PM2.5, 42%; PM2.5 absorbance, 67%). CONCLUSIONS: Using a large data set from 23 European study areas, we were able to develop LUR models for NO2 and PM metrics that predicted measurements made at independent sites and areas reasonably well. This finding is useful for assessing exposure in health studies conducted in areas where no measurements were conducted.

Development of land use regression models for particle composition in twenty study areas in Europe.

Land Use Regression (LUR) models have been used to describe and model spatial variability of annual mean concentrations of traffic related pollutants such as nitrogen dioxide (NO2), nitrogen oxides (NOx) and particulate matter (PM). No models have yet been published of elemental composition. As part of the ESCAPE project, we measured the elemental composition in both the PM10 and PM2.5 fraction sizes at 20 sites in each of 20 study areas across Europe. LUR models for eight a priori selected elements (copper (Cu), iron (Fe), potassium (K), nickel (Ni), sulfur (S), silicon (Si), vanadium (V), and zinc (Zn)) were developed. Good models were developed for Cu, Fe, and Zn in both fractions (PM10 and PM2.5) explaining on average between 67 and 79% of the concentration variance (R(2)) with a large variability between areas. Traffic variables were the dominant predictors, reflecting nontailpipe emissions. Models for V and S in the PM10 and PM2.5 fractions and Si, Ni, and K in the PM10 fraction performed moderately with R(2) ranging from 50 to 61%. Si, NI, and K models for PM2.5 performed poorest with R(2) under 50%. The LUR models are used to estimate exposures to elemental composition in the health studies involved in ESCAPE.

Evaluation of land use regression models for NO2 and particulate matter in 20 European study areas: the ESCAPE project.

Land use regression models (LUR) frequently use leave-one-out-cross-validation (LOOCV) to assess model fit, but recent studies suggested that this may overestimate predictive ability in independent data sets. Our aim was to evaluate LUR models for nitrogen dioxide (NO2) and particulate matter (PM) components exploiting the high correlation between concentrations of PM metrics and NO2. LUR models have been developed for NO2, PM2.5 absorbance, and copper (Cu) in PM10 based on 20 sites in each of the 20 study areas of the ESCAPE project. Models were evaluated with LOOCV and "hold-out evaluation (HEV)" using the correlation of predicted NO2 or PM concentrations with measured NO2 concentrations at the 20 additional NO2 sites in each area. For NO2, PM2.5 absorbance and PM10 Cu, the median LOOCV R(2)s were 0.83, 0.81, and 0.76 whereas the median HEV R(2) were 0.52, 0.44, and 0.40. There was a positive association between the LOOCV R(2) and HEV R(2) for PM2.5 absorbance and PM10 Cu. Our results confirm that the predictive ability of LUR models based on relatively small training sets is overestimated by the LOOCV R(2)s. Nevertheless, in most areas LUR models still explained a substantial fraction of the variation of concentrations measured at independent sites.

Ambient air pollution and low birthweight: a European cohort study (ESCAPE).

BACKGROUND: Ambient air pollution has been associated with restricted fetal growth, which is linked with adverse respiratory health in childhood. We assessed the effect of maternal exposure to low concentrations of ambient air pollution on birthweight. METHODS: We pooled data from 14 population-based mother-child cohort studies in 12 European countries. Overall, the study population included 74 178 women who had singleton deliveries between Feb 11, 1994, and June 2, 2011, and for whom information about infant birthweight, gestational age, and sex was available. The primary outcome of interest was low birthweight at term (weight <2500 g at birth after 37 weeks of gestation). Mean concentrations of particulate matter with an aerodynamic diameter of less than 2·5 µm (PM2·5), less than 10 µm (PM10), and between 2·5 µm and 10 µm during pregnancy were estimated at maternal home addresses with temporally adjusted land-use regression models, as was PM2·5 absorbance and concentrations of nitrogen dioxide (NO2) and nitrogen oxides. We also investigated traffic density on the nearest road and total traffic load. We calculated pooled effect estimates with random-effects models. FINDINGS: A 5 µg/m(3) increase in concentration of PM2·5 during pregnancy was associated with an increased risk of low birthweight at term (adjusted odds ratio [OR] 1·18, 95% CI 1·06-1·33). An increased risk was also recorded for pregnancy concentrations lower than the present European Union annual PM2·5 limit of 25 µg/m(3) (OR for 5 µg/m(3) increase in participants exposed to concentrations of less than 20 µg/m(3) 1·41, 95% CI 1·20-1·65). PM10 (OR for 10 µg/m(3) increase 1·16, 95% CI 1·00-1·35), NO2 (OR for 10 µg/m(3) increase 1·09, 1·00-1·19), and traffic density on nearest street (OR for increase of 5000 vehicles per day 1·06, 1·01-1·11) were also associated with increased risk of low birthweight at term. The population attributable risk estimated for a reduction in PM2·5 concentration to 10 µg/m(3) during pregnancy corresponded to a decrease of 22% (95% CI 8-33%) in cases of low birthweight at term. INTERPRETATION: Exposure to ambient air pollutants and traffic during pregnancy is associated with restricted fetal growth. A substantial proportion of cases of low birthweight at term could be prevented in Europe if urban air pollution was reduced. FUNDING: The European Union.

Development of Land Use Regression models for PM(2.5), PM(2.5) absorbance, PM(10) and PM(coarse) in 20 European study areas; results of the ESCAPE project.

Land Use Regression (LUR) models have been used increasingly for modeling small-scale spatial variation in air pollution concentrations and estimating individual exposure for participants of cohort studies. Within the ESCAPE project, concentrations of PM(2.5), PM(2.5) absorbance, PM(10), and PM(coarse) were measured in 20 European study areas at 20 sites per area. GIS-derived predictor variables (e.g., traffic intensity, population, and land-use) were evaluated to model spatial variation of annual average concentrations for each study area. The median model explained variance (R(2)) was 71% for PM(2.5) (range across study areas 35-94%). Model R(2) was higher for PM(2.5) absorbance (median 89%, range 56-97%) and lower for PM(coarse) (median 68%, range 32- 81%). Models included between two and five predictor variables, with various traffic indicators as the most common predictors. Lower R(2) was related to small concentration variability or limited availability of predictor variables, especially traffic intensity. Cross validation R(2) results were on average 8-11% lower than model R(2). Careful selection of monitoring sites, examination of influential observations and skewed variable distributions were essential for developing stable LUR models. The final LUR models are used to estimate air pollution concentrations at the home addresses of participants in the health studies involved in ESCAPE.

[Closed femoral fracture complicated by blunt injury and posttraumatic occlusion of the superficial femoral artery--a case review]. / Zatvorená zlomenina femoru komplikovaná tupým poranením a poúrazovou oklúziou a. femoralis superficialis--kazuistika.

The diagnosis and the treatment of an unusual posttraumatic occlusion of a femoral superficial artery during skeletal traction has been described. Angiography of the arteries of lower extremities was done because a sharp pain, cold, parestesia of the foot and absent pulsies had been found. The occlusion of the right femoral superficial artery was seen in the place of the compression of the artery by a fragment of femur. The fracture of the femur was stabilized with Ender's rods immediately and the injured part of the artery was resected and replaced by a venous graft. The healing was without any complication. The symptoms, the diagnostic procedures and the therapy of the occlusion of the arteries caused by fractures of the extremities has been discussed.

Replacement of Fab-7 by the gypsy or scs insulator disrupts long-distance regulatory interactions in the Abd-B gene of the bithorax complex.

Chromatin domain boundaries, like scs or gypsy insulators in Drosophila, have been identified in transgene assays through their enhancer-blocking activity. Boundary elements in the bithorax complex (BX-C), such as Fab-7 and Fab-8, have been identified genetically and been shown to have insulator activity in transgene assays. However, it is not clear whether boundary elements identified in transgene assays will function appropriately in chromosomal contexts such as BX-C. Using gene conversion, we have substituted the scs or gypsy insulators for Fab-7. We find that both scs and gypsy are very potent insulators in the ectoderm, but surprisingly, the insulating activity of gypsy (but not scs) is lost in the CNS. Our results reveal that the Fab-7 boundary must have special properties that scs and gypsy lack, which allow it to function appropriately in BX-C regulation.

Variations of components of the plasminogen activation system with the cell cycle in benign prostate tissue and prostate cancer.

BACKGROUND: Components of the fibrinolytic system are involved in tumor cell invasion and metastasis. Previous investigations suggested a cell cycle-dependent expression of urokinase-type plasminogen activator (u-PA) in epithelial cells. In order to determine a correlation of cell cycle phases with the fibrinolytic system, we investigated the expression of u-PA, tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor type 1 (PAI-1) in normal and tumor-containing prostate extracts and analyzed a possible relationship with flow cytometry-determined proliferative activity of the samples. Cell cycle phases were correlated with fibrinolytic parameters in prostate tissue. METHODS: Samples were obtained from patients undergoing radical prostatectomy for prostate cancer and separated into two portions for DNA analysis and the detection of u-PA, t-PA, and PAI-1. Flow cytometric analysis was performed according to the Vindelov technique. The concentrations of u-PA, t-PA, and PAI-1 were determined from tissue extracts after homogenization by an enzyme-linked immunosorbent assay (ELISA) technique. RESULTS: Correlations of u-PA and t-PA expression with the frequency of G0/G1, S, G2M, S-phase fraction (SPF), and proliferation index (PI) for normal prostate and prostate cancer revealed no significant correlation. The only significant finding was observed in normal tissue revealing a positive correlation between PAI-1 expression and G0/G1 and a negative correlation with S-phase, SPF, and PI. No dependence of PAI-1 expression on different cell phases was found in prostate cancer. Furthermore, no significant correlation of u-PA, t-PA, and PAI-1 with cell cycles in organ-confined ( or = pT3a) tumors was found. No significant correlation in prostate cancer of components of the fibrinolytic system differentiated according to tumor grade or perineural tumor infiltration and cell cycle analysis was found. Only in highly differentiated G1 (Gleason 2-4) cancer, u-PA had a significant positive correlation with G2M-phase. CONCLUSION: Absence of a correlation between levels of components of the fibrinolytic system and cell cycle phases suggests that the reported association between increases of some of these components and aggressive biological behavior of prostate cancer is secondary to non-cell cycle-related mechanisms.

The role of the Drosophila TAK homologue dTAK during development.

The TAK kinases belong to the MAPKKK group and have been implicated in a variety of signaling events. Originally described as a TGF-beta activated kinase (TAK) it has, however, subsequently been demonstrated to signal through p38, Jun N-terminal kinase (JNK) and Nemo types of MAP kinases, and the NFkappaB inducing kinase. Despite these multiple proposed functions, the in vivo role of TAK family kinases remains unclear. Here we report the isolation and genetic characterization of the Drosophila TAK homologue (dTAK). By employing overexpression and double-stranded RNA interference (RNAi) techniques we have analyzed its function during embryogenesis and larval development. Overexpression of dTAK in the embryonic epidermis is sufficient to induce the transcription of the JNK target genes decapentaplegic and puckered. Furthermore, overexpression of dominant negative (DN) or wild-type forms of dTAK in wing and eye imaginal discs, respectively, results in defects in thorax closure and ommatidial planar polarity, two well described phenotypes associated with JNK signaling activity. Surprisingly, RNAi and DN-dTAK expression studies in the embryo argue for a differential requirement of dTAK during developmental processes controlled by JNK signaling, and a redundant or minor role of dTAK in dorsal closure. In addition, dTAK-mediated activation of JNK in the Drosophila eye imaginal disc leads to an eye ablation phenotype due to ectopically induced apoptotic cell death. Genetic analyses in the eye indicate that dTAK can also act through the p38 and Nemo kinases in imaginal discs. Our results suggest that dTAK can act as a JNKKK upstream of JNK in multiple contexts and also other MAPKs in the eye. However, the loss-of-function RNAi studies indicate that it is not strictly required and thus either redundant or playing only a minor role in the context of embryonic dorsal closure.

The iab-7 polycomb response element maps to a nucleosome-free region of chromatin and requires both GAGA and pleiohomeotic for silencing activity.

In the work reported here we have undertaken a functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila melanogaster bithorax complex (BX-C). Previous studies mapped the iab-7 PRE to an 860-bp fragment located just distal to the Fab-7 boundary. Located within this fragment is an approximately 230-bp chromatin-specific nuclease-hypersensitive region called HS3. We have shown that HS3 is capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of a mini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. We show that GAGA and Pho interact with these sequences in vitro and that the consensus binding sites for the two proteins are critical for the silencing activity of the iab-7 PRE in vivo.

The Ketel gene encodes a Drosophila homologue of importin-beta.

The Drosophila melanogaster Ketel gene was identified via the Ketel(D) dominant female sterile mutations and their ketel(r) revertant alleles that are recessive zygotic lethals. The maternally acting Ketel(D) mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1. 2-3 in four ketel(r) alleles. The Ketel(+) transgenes rescue ketel(r)-associated zygotic lethality and slightly reduce Ketel(D)-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-beta, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm from stage 11 of oogenesis. In cleavage embryos the Ketel protein is cytoplasmic. The Ketel gene appears to be ubiquitously expressed in embryonic cells. Western blot analysis revealed that the Ketel gene is not expressed in several larval cell types of late third instar larvae.

Signaling specificity by Frizzled receptors in Drosophila.

Wnt-Frizzled (Fz) signaling pathways play recurring important roles during the development and homeostasis of vertebrates and invertebrates. Fz receptors can signal through beta-catenin-dependent and -independent pathways. In Drosophila, Fz and Fz2 are redundant receptors for Wg. In addition, Fz conveys signals through a distinct pathway to organize planar polarization of epithelial structures. We demonstrate that the cytoplasmic sequences of Fz2 and Fz preferentially activate the beta-catenin and planar polarity cascade, respectively. Both receptors activate either pathway, but with different efficiencies. Intrinsic differences in signaling efficiency in closely related receptors might be a general mechanism for generating signaling specificity in vivo.

Involvement of low-density lipoprotein receptor-related protein (LRP) in the clearance of factor VIII in von Willebrand factor-deficient mice.

Factor VIII is tightly noncovalently linked to von Willebrand factor (vWF) in plasma with a stoichiometry of 1:50, and vWF deficiency results in secondary factor VIII deficiency, with accelerated clearance of factor VIII from the circulation. We used a murine model of severe von Willebrand disease (vWF knockout mice) to study the effect of a recombinant vWF/pro-vWF preparation (rpvWF) on factor VIII survival and to investigate whether low-density lipoprotein receptor-related protein (LRP) might be involved in the in vivo clearance of factor VIII in the absence of vWF. vWF-deficient mice received 70 U/kg rpvWF in the first series of experiments, and in a second series, 80 mg/kg receptor-associated protein (RAP) as a recombinant fusion protein to block the action of LRP. Factor VIII levels were measured at time 0, or 1 or 3 hours after administration of rpvWF or RAP. RAP induced a sustained rise in factor VIII levels comparable to that induced by rpvWF. In a third series, the preadministration of RAP resulted in a slower disappearance of factor VIII antigen (measured by an enzyme-linked immunosorbent assay specific for human factor VIII) after infusion of recombinant factor VIII. These findings suggest that the accelerated clearance of factor VIII seen in the absence of vWF may be a result of the involvement of LRP in factor VIII metabolism. (Blood. 2000;95:1703-1708)

The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain.

The Drosophila bithorax complex Abdominal-B (Abd-B) gene specifies parasegmental identity at the posterior end of the fly. The specific pattern of Abd-B expression in each parasegment (PS) determines its identity and, in PS10-13, Abd-B expression is controlled by four parasegment-specific cis-regulatory domains, iab-5 to iab-8, respectively. In order to properly determine parasegmental identity, these four cis-regulatory domains must function autonomously during both the initiation and maintenance phases of BX-C regulation. The studies reported here demonstrate that the (centromere) distal end of iab-7 domain is delimited by the Fab-8 boundary. Initiators that specify PS12 identity are located on the proximal iab-7 side of Fab-8, while initiators that specify PS13 identity are located on the distal side of Fab-8, in iab-8. We use transgene assays to demonstrate that Fab-8 has enhancer blocking activity and that it can insulate reporter constructs from the regulatory action of the iab-7 and iab-8 initiators. We also show that the Fab-8 boundary defines the realm of action of a nearby iab-8 Polycomb Response Element, preventing this element from ectopically silencing the adjacent domain. Finally, we demonstrate that the insulating activity of the Fab-8 boundary in BX-C is absolutely essential for the proper specification of parasegmental identity by the iab-7 and iab-8 cis-regulatory domains. Fab-8 together with the previously identified Fab-7 boundary delimit the first genetically defined higher order domain in a multicellular eukaryote.