The Effect of Surface Pretreatment and Water Storage on the Bonding Strength of a Resin Composite Cement to Modified PEEK.

The bonding quality of bonding to polyether ether ketone (PEEK) after different surface treatments and adhesive regimens was assessed through shear bond strength. MATERIALS AND METHODS: Sixty modified PEEK disks were cut out of BioHPP round blanks using CAD-CAM procedures. Disks were subjected to the following surface pretreatments: (A) Sandblasting with alumina (Rocatec) and application of adhesive bonding agent visiolink (control group) (B) Sandblasting with silica-modified alumina (Cojet), application of silane agent Espe Sil, followed by application of adhesive bonding agent Visio-Bond (C) Sandblasting with silica-modified alumina (Cojet), application of silane containing primer-adhesive Clearfil Ceramic Primer, followed by application of adhesive bonding agent visiolink. A dual-curing resin composite cement (combolign) was luted to all treated surfaces. Each group was further divided to subgroups of 10 specimens which were stored in distilled water at 37°C for 150 days without further thermocycling. Specimens were then submitted to shear bond strength testing. RESULTS: Group (B) was statistically significant different from group (A) (control group). Water storage condition had no significant influence on final bond strength. CONCLUSIONS: Use of different conditioning protocols had a significant effect on the final bond strength of composite resin cement to PEEK surface. Water storage did not significantly influence bonding.

Safety evaluation of mechanical recycling processes used to produce polyethylene terephthalate (PET) intended for food contact applications.

The development of a scheme for the safety evaluation of mechanical recycling processes for polyethylene terephthalate (PET) is described. The starting point is the adoption of a threshold of toxicological concern such that migration from the recycled PET should not give rise to a dietary exposure exceeding 0.0025 µg kg(-1) bw day(-1), the exposure threshold value for chemicals with structural alerts raising concern for potential genotoxicity, below which the risk to human health would be negligible. It is practically impossible to test every batch of incoming recovered PET and every production batch of recycled PET for all the different chemical contaminants that could theoretically arise. Consequently, the principle of the safety evaluation is to measure the cleaning efficiency of a recycling process by using a challenge test with surrogate contaminants. This cleaning efficiency is then applied to reduce a reference contamination level for post-consumer PET, conservatively set at 3 mg kg(-1) PET for a contaminant resulting from possible misuse by consumers. The resulting residual concentration of each contaminant in recycled PET is used in conservative migration models to calculate migration levels, which are then used along with food consumption data to give estimates of potential dietary exposure. The default scenario, when the recycled PET is intended for general use, is that of an infant weighing 5 kg and consuming every day powdered infant formula reconstituted with 0.75 L of water coming from water bottles manufactured with 100% recycled PET. According to this scenario, it can be derived that the highest concentration of a substance in water that would ensure that the dietary exposure of 0.0025 µg kg(-1) bw day(-1) is not exceeded, is 0.017 µg kg(-1) food. The maximum residual content that would comply with this migration limit depends on molecular weight and is in the range 0.09-0.32 mg kg(-1) PET for the typical surrogate contaminants.

Differential mammary morphogenesis along the anteroposterior axis in Hoxc6 gene targeted mice.

Mammary epithelial cell proliferation, branching, and differentiation span from the appearance of the mammary bud in midgestation through to the cycling mammary gland in adulthood. Here, we show that females homozygous for a targeted disruption of the Hoxc6 homeobox gene produce thoracic mammary glands that are slightly under-developed at birth and completely cleared of epithelium by adulthood, and inguinal mammary ducts that are dilated and fail to regress in response to ovariectomy. Mammary buds are detected in E12.5 Hoxc6 homozygous embryos. However, in newborn Hoxc6 homozygous females, branching ductal structures and fat pad development are reduced. Whole-mount and histologic analyses of mammary glands from adult Hoxc6 homozygous females show the absence of mammary epithelium in thoracic glands and dilated ducts in inguinal glands at 100% penetrance. Histologic analysis of inguinal mammary glands from ovariectomized Hoxc6 homozygous females demonstrates no signs of the expected regression of epithelium, suggesting that these glands are not responsive to the loss of ovarian hormone signals. We further observe repression of Hoxc6 expression specifically within mammary stroma by estrogen and progesterone. Hoxc6 homozygous mice also exhibit a homeotic transformation of the second thoracic vertebra into the first (T2 to T1 conversion with 60% penetrance), corresponding to both the gene's anterior boundary of expression and the most extreme appearance of mammary defects. The position-specific phenotypes observed and the potential role for Hoxc6 in mediating hormone-regulated ductal expansion and regression in the adult female are discussed.

Control of neurulation by the nucleosome assembly protein-1-like 2.

Neurulation is a complex process of histogenesis involving the precise temporal and spatial organization of gene expression. Genes influencing neurulation include proneural genes determining primary cell fate, neurogenic genes involved in lateral inhibition pathways and genes controlling the frequency of mitotic events. This is reflected in the aetiology and genetics of human and mouse neural tube defects, which are of both multifactorial and multigenic origin. The X-linked gene Nap1l2, specifically expressed in neurons, encodes a protein that is highly similar to the nucleosome assembly (NAP) and SET proteins. We inactivated Nap1l2 in mice by gene targeting, leading to embryonic lethality from mid-gestation onwards. Surviving mutant chimaeric embryos showed extensive surface ectoderm defects as well as the presence of open neural tubes and exposed brains similar to those observed in human spina bifida and anencephaly. These defects correlated with an overproduction of neuronal precursor cells. Protein expression studies showed that the Nap1l2 protein binds to condensing chromatin during S phase and in apoptotic cells, but remained cytoplasmic during G1 phase. Nap1l2 therefore likely represents a class of tissue-specific factors interacting with chromatin to regulate neuronal cell proliferation.

Hemorrhage, impaired hematopoiesis, and lethality in mouse embryos carrying a targeted disruption of the Fli1 transcription factor.

The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis.

Mouse models in the study of the Ets family of transcription factors.

The Ets family of transcription factors is one of a growing number of master regulators of development. This family was originally defined by the presence of a conserved DNA binding domain, the Ets domain. To date, nearly 30 members of this family have been identified and implicated in a wide range of physiological and pathological processes. Despite the likely importance of Ets-family members, each of their precise roles has not been delineated. Herein, we describe the elucidation of essential functions of a few of these family members in vivo using knockout mouse models. Of the knockouts generated to date, the majority shows important functions in hematopoiesis, ranging from PU.1, a principle regulator of myelo-lymphopoiesis, to Spi-B which regulates the proper function of terminally differentiated cells. Ets1 was shown to be of intermediate importance as a regulator of pan-lymphoid development. Other Ets family members such as Fli1 and TEL1 display distinct and/or overlapping functions in vasculo/angiogenesis, hemostasis and hematopoiesis. The remaining knockouts generated, Ets2 and Er81, show non-hematopoietic defects related to extraembryonic development and neurogenesis, respectively. The pioneering group of knockout models described reveals only the most distinct functions of each of these Ets family members. A better understanding of the roles and hierarchies of Ets family members in cellular differentiation will come with the generation of new null alleles in previously untargeted family members, more mutant alleles in members already disrupted, double knockouts, ES cell differentiation and chimera rescue experiments, and tissue-specific inducible knockouts.

Targeted disruption of Hoxd9 and Hoxd10 alters locomotor behavior, vertebral identity, and peripheral nervous system development.

The five most 5' HoxD genes, which are related to the Drosophila Abd-B gene, play an important role in patterning axial and appendicular skeletal elements and the nervous system of developing vertebrate embryos. Three of these genes, Hoxd11, Hoxd12, and Hoxd13, act synergistically to pattern the hindlimb autopod. In this study, we examine the combined effects of two additional 5' HoxD genes, Hoxd9 and Hoxd10. Both of these genes are expressed posteriorly in overlapping domains in the developing neural tube and axial mesoderm as well as in developing limbs. Locomotor behavior in animals carrying a double mutation in these two genes was altered; these alterations included changes in gait, mobility, and adduction. Morphological analysis showed alterations in axial and appendicular skeletal structure, hindlimb peripheral nerve organization and projection, and distal hindlimb musculature. These morphological alterations are likely to provide the substrate for the observed alterations in locomotor behavior. The alterations observed in double-mutant mice are distinct from the phenotypes observed in mice carrying single mutations in either gene, but exhibit most of the features of both individual phenotypes. This suggests that the combined activity of two adjacent Hox genes provides more patterning information than activity of each gene alone. These observations support the idea that adjacent Hox genes with overlapping expression patterns may interact functionally to provide patterning information to the same regions of developing mouse embryos.

Stability testing of the plastics additives 2,4-dihydroxybenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone, 2-hydroxy-4-n-octyloxybenzophenone, 2-ethoxy-2-ethyloxanilide (tinuvin 312) and 2,2'-methylenebis(4-methyl-t-tert-butyl phenol) in aqueous and fatty food simulants.

The stability of five additives used in the manufacture of plastics materials intended to be in contact with foodstuffs was tested in two EU aqueous food simulants (3% acidic acid and 15% ethanol), in olive oil and in two alternative fatty food simulants (95% ethanol and isooctane) under various conditions of temperature and time established for migration testing. The additives were the following: 2,4-dihydroxybenzophenone, 2-2'-dihydroxy-4-methoxybenzophenone, 2-hydroxy-4-n-octyloxybenzophenone, 2-ethoxy-2-ethyloxanilide and 2,2'-methylenebis(4-methyl-6-tert-butyl phenol). The additives were spiked into the simulants at their anticipated Specific Migration Limit (SML) and submitted to time-temperature conditions used in the migration testing. The test samples were analysed against fresh samples of the additives spiked at the same concentration just before the analysis. All samples were analysed with HPLC, using an ODS2 column and mobile phase of methanol-water or acetonitrile-water. The results were statistically evaluated by applying a two-tailed F-test (at 95% confidence level) to the variances and onetailed t-test (at 90% confidence level) to the means of the control and the test samples. The statistical evaluation of the results showed that the additives were stable in all simulants within the statistical allowances. Only the 2.2'-methylenebis(4-methyl-6-tert-butyl phenol) showed a loss of about 20% in olive oil and 17% in 3% aqueous acetic acid. The results of the study will be used by the Commission, DG III-C-I. to help to determine whether legislative restrictions in the additives Directive should be applied to the finished material or article (QM) or to migration to simulants (SML).

Targeted disruption of the even-skipped gene, evx1, causes early postimplantation lethality of the mouse conceptus.

Implantation within the mammalian uterus elicits dramatic changes in the growth, differentiation, and morphogenesis of the conceptus. This process is interrupted in mice carrying a targeted disruption of the murine evx1 gene, a homolog of the Drosophila even-skipped (eve) gene. Upon implantation, presumptive evx1- homozygotes elicit a decidual response, invade the uterine epithelium, and attach to the basement membrane between uterine stroma and epithelium, but fail to differentiate extraembryonic tissues or to form egg cylinders prior to resorption. Retrograde analysis of embryo genotypes demonstrates that homozygotes could be isolated as free-floating blastocysts but not as gastrulating egg cylinders. Homozygous mutant blastocysts appeared normal and, when grown in vitro, attach, proliferate, and form trophoblastic giant cells surrounding a growing inner cell mass before rapidly degenerating. In situ hybridization analysis demonstrates evx1 gene expression within the visceral endoderm after implantation and prior to gastrulation, at a time in which the mutant phenotype is first detected.

Utilization of DNA recombination for the two-step replacement of growth factor sequences in the vaccinia virus genome.

An efficient procedure for the generation of sequence-specific alterations of the vaccinia virus genome was demonstrated. Homologous DNA recombination within cells infected with vaccinia virus was used for the deletion or replacement of promoter sequences of the viral growth factor gene by a procedure comparable to transplacement in Saccharomyces cerevisiae. This DNA replacement procedure can potentially be used to generate any sequence alteration within the vaccinia virus genome. Deletion of growth factor promoter sequences resulted in a dramatic reduction in growth factor gene transcription and protein synthesis. Replacement of growth factor promoter sequences with promoter sequences of the strong constitutive 40-kDa gene resulted in an increase in gene transcription and protein synthesis and an altered temporal pattern of expression. Virus containing mutations in the growth factor gene demonstrated different plaque morphologies on cell culture monolayers.

Delineation of the viral products of recombination in vaccinia virus-infected cells.

Plasmids containing the vaccinia virus thymidine kinase gene, its flanking DNA sequences, and the Escherichia coli beta-galactosidase gene were used in conjunction with a thymidine kinase-deficient virus to examine the viral products of recombination. Progeny derived from single-crossover events could be distinguished from those generated by gene conversion or double-crossover events when the beta-galactosidase gene was separated from the thymidine kinase gene by the flanking sequences. Using methotrexate to select for recombinant virus and a chromogenic indicator to detect beta-galactosidase, the generation of viral recombinants was measured over a 48-h period. Recombinant progeny were first observed at 12 h and increased to a maximum of 2.5% at 48 h. Single-crossover products, as determined by beta-galactosidase expression, reached a maximum of 57% of the recombinant population at 24 h and thereafter declined. DNA hybridization analysis was used to examine genomic structures of the progeny of the initial viral plaques, plaques purified three times, and those subject to a 10(4)-fold amplification. These analyses confirmed that single-crossover events within either the 5'- or 3'-homologous flanking sequences generated unstable recombinant structures. These structures were shown to contain a single copy of the intact thymidine kinase gene within the corresponding copy of the duplicated thymidine kinase flanking sequences, separated by the beta-galactosidase gene and plasmid DNA. Significantly, these duplicated structures could undergo further recombination to produce repeats of either the intact or the deleted thymidine kinase sequences. These intermediate structures ultimately degenerated to produce either the parental thymidine kinase-deleted or the wild-type genome. The wild-type genome was also shown to be generated directly by gene conversion or double-crossover events.

The transforming sequences of avian myelocytomatosis virus (MC29).

Avian myelocytomatosis virus (MC29), a defective acute leukemia virus, has a broad oncogenic spectrum in vivo, and transforms fibroblasts and hematopoietic target cells in vitro, We have used recombinant DNA technology to isolate and characterize the sequences that are essential in the transformation process. Integrated MC29 proviral DNA was isolated from a library of recombinant phage containing DNA from the MC29-transformed nonproducer quail cell line Q5. The cloned DNA was analyzed by Southern blotting of restriction endonuclease digests and by electron microscopic visualization of R-loops formed between the cloned DNA and MC29 or helper virus RNA. It was found that the 9.2 kb cloned DNA insert contains approximately 4 kb of viral sequences and 5.2 kb of quail cellular sequences. The viral sequences contain all of the MC29 specific sequences and 5' helper related sequences as well as part of the envelope region. The size of the cloned EcoRI fragment is the same as that of the major band in EcoRI-cleaved Q5 DNA that hybridizes to viral sequences. Transfection of the cloned DNA into NIH 3T3 cells revealed that the MC29-specific sequences are functional in that they induce foci of transformed cells with high efficiency.