Highly efficient germ-line transmission of proviral insertions in zebrafish.

An important technology in model organisms is the ability to make transgenic animals. In the past, transgenic technology in zebrafish has been limited by the relatively low efficiency with which transgenes could be generated using either DNA microinjection or retroviral infection. Previous efforts to generate transgenic zebrafish with retroviral vectors used a pseudotyped virus with a genome based on the Moloney murine leukemia virus and the envelope protein of the vesicular stomatitis virus. This virus was injected into blastula-stage zebrafish, and 16% of the injected embryos transmitted proviral insertions to their offspring, with most founders transmitting a single insertion to approximately 2% of their progeny. In an effort to improve this transgenic frequency, we have generated pseudotyped viral stocks of two new Moloney-based genomes. These viral stocks have titers up to two orders of magnitude higher than that used previously. Injection of these viruses resulted in a dramatic increase in transgenic efficiency; over three different experiments, 83% (110/133) of the injected embryos transmitted proviral insertions to 24% of their offspring. Furthermore, founders made with one of the viruses transmitted an average of 11 different insertions through their germ line. These results represent a 50- to 100-fold improvement in the efficiency of generating transgenic zebrafish, making it now feasible for a single lab to rapidly generate tens to hundreds of thousands of transgenes. Consequently, large-scale insertional mutagenesis strategies, previously limited to invertebrates, may now be possible in a vertebrate.

Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96/GRP94.

Heat shock protein gp96 primes class I restricted cytotoxic T cells against antigens present in the cells from which it was isolated. Moreover, gp96 derived from certain tumors functions as an effective vaccine, causing complete tumor regressions in in vivo tumor challenge protocols. Because tumor-derived gp96 did not differ from gp96 isolated from normal tissues, a role for gp96 as a peptide carrier has been proposed. To test this hypothesis, we analyzed whether such an association of antigenic peptides with gp96 occurs in a well-defined viral model system. Here we present the full characterization of an antigenic peptide that endogenously associates with the stress protein gp96 in cells infected with vesicular stomatitis virus (VSV). This peptide is identical to the immunodominant peptide of VSV, which is also naturally presented by H-2Kb major histocompatibility complex class I molecules. This peptide associates with gp96 in VSV-infected cells regardless of the major histocompatibility com- plex haplotype of the cell. Our observations provide a biochemical basis for the vaccine function of gp96.

Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.

Production of transgenic dwarf surfclams, Mulinia lateralis, with pantropic retroviral vectors.

A pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus was used as a gene transfer vector in the dwarf surfclam, Mulinia lateralis. These pantropic retroviral vectors have an extremely broad host cell range and can infect many nonmammalian species. Newly fertilized dwarf surfclam eggs were electroporated at 700 V in the presence of 1 x 10(4) colony-forming units of pantropic pseudotyped retroviral particles. Infection was well tolerated and did not affect the survival rate of the embryos. Gametes collected from P1 presumptive transgenic animals were analyzed for the presence of provirus by PCR, and in different experiments 13-33% of the gamete pools were positive for the transgene. Dot blot hybridization of DNA samples from the F1 offspring of two different crosses between infected P1 and wild-type individuals revealed that 28% and 31% of F1 offspring were transgenic, respectively. Southern blot analysis of DNA isolated from PCR-positive F1 animals confirmed integration of a single copy of the provirus into the host genome. Thus, the germ lines of these two P1 transgenic animals were mosaic for the transgene. Expression of beta-galactosidase encoded by the provirus was detected in transgenic but not control surfclam embryos. Pantropic pseudotyped retroviral vectors provide a useful method for the stable introduction of foreign genetic information into surfclams and may facilitate the introduction of desirable genetic traits into commercially important shellfish and crustaceans.

Inhibition of human immunodeficiency virus type 1 and vaccinia virus infection by a dominant negative factor of the interferon regulatory factor family expressed in monocytic cells.

ICSBP is a member of the interferon (IFN) regulatory factor (IRF) family that regulates expression of type I interferon (IFN) and IFN-regulated genes. To study the role of the IRF family in viral infection, a cDNA for the DNA-binding domain (DBD) of ICSBP was stably transfected into U937 human monocytic cells. Clones that expressed DBD exhibited a dominant negative phenotype and did not elicit antiviral activity against vesicular stomatitis virus (VSV) infection upon IFN treatment. Most notably, cells expressing DBD were refractory to infection by vaccinia virus (VV) and human immunodeficiency virus type 1 (HIV-1). The inhibition of VV infection was attributed to defective virion assembly, and that of HIV-1 to low CD4 expression and inhibition of viral transcription in DBD clones. HIV-1 and VV were found to have sequences in their regulatory regions similar to the IFN-stimulated response element (ISRE) to which IRF family proteins bind. Accordingly, these viral sequences and a cellular ISRE bound a shared factor(s) expressed in U937 cells. These observations suggest a novel host-virus relationship in which the productive infection of some viruses is regulated by the IRF-dependent transcription pathway through the ISRE.

Does immediate loading affect clinical and patient-centered outcomes of mandibular 2-unsplinted-implant overdenture? : a 2-year within-case analysis

Objective To provide 2-year clinical- and patient-oriented data with regard to mandibular overdenture assisted by 2 immediately loaded unsplinted implants. Material and methods In this pre-post design, Phase-I clinical trial, 18 edentate individuals (62.4 ± 7.7 years) received a new set of complete denture. Then, following standard procedures, 3 threaded implants (OsseoSpeed TX™, Dentsply Implants, Mölndal, Sweden) were placed in the mandible in each patient, and locator abutments (Zest Anchors LLC, Escondido, U.S.A.) were inserted on the right and left side implants. The midline implant served as a control for within-patient comparison. The immediate loading was conducted within 24 h of surgery. Data were collected at baseline (T0), 12 (T1) and 24 (T2) months after immediate loading. The clinical outcomes included implant survival rate, crestal bone level changes and implant stability. These criteria were assessed through clinical and radiographic examinations as well as resonance frequency analysis. Patient-centered outcomes included patient satisfaction and oral health-related quality of life measured using validated questionnaires. Brunner-Langer approach was used for statistical analysis. Results Implant survival rate for immediate loaded implants was 91.7% at 2-year follow-up. None of the unloaded implants failed. There was no statistically significant difference at baseline and follow-ups with regard to clinical outcomes between loaded and unloaded implants. Patient satisfaction and quality of life improved (p < 0.0001) from baseline to 2-year follow-up. Conclusion Immediate loading protocol did not negatively affect clinical outcomes, satisfaction and quality of life of patients wearing 2-unsplinted-implant mandibular overdenture for 2 years. This conclusion requires confirmation by randomized control trials. Clinical significance statement Mandibular overdenture assisted by two immediately-loaded unsplinted implants is successful treatment based on 2-year clinical and patient-based outcomes.

Does immediate loading affect clinical and patient-centered outcomes of mandibular 2-unsplinted-implant overdenture? : a 2-year within-case analysis

Objective To provide 2-year clinical- and patient-oriented data with regard to mandibular overdenture assisted by 2 immediately loaded unsplinted implants. Material and methods In this pre-post design, Phase-I clinical trial, 18 edentate individuals (62.4 ± 7.7 years) received a new set of complete denture. Then, following standard procedures, 3 threaded implants (OsseoSpeed TX™, Dentsply Implants, Mölndal, Sweden) were placed in the mandible in each patient, and locator abutments (Zest Anchors LLC, Escondido, U.S.A.) were inserted on the right and left side implants. The midline implant served as a control for within-patient comparison. The immediate loading was conducted within 24 h of surgery. Data were collected at baseline (T0), 12 (T1) and 24 (T2) months after immediate loading. The clinical outcomes included implant survival rate, crestal bone level changes and implant stability. These criteria were assessed through clinical and radiographic examinations as well as resonance frequency analysis. Patient-centered outcomes included patient satisfaction and oral health-related quality of life measured using validated questionnaires. Brunner-Langer approach was used for statistical analysis. Results Implant survival rate for immediate loaded implants was 91.7% at 2-year follow-up. None of the unloaded implants failed. There was no statistically significant difference at baseline and follow-ups with regard to clinical outcomes between loaded and unloaded implants. Patient satisfaction and quality of life improved (p < 0.0001) from baseline to 2-year follow-up. Conclusion Immediate loading protocol did not negatively affect clinical outcomes, satisfaction and quality of life of patients wearing 2-unsplinted-implant mandibular overdenture for 2 years. This conclusion requires confirmation by randomized control trials. Clinical significance statement Mandibular overdenture assisted by two immediately-loaded unsplinted implants is successful treatment based on 2-year clinical and patient-based outcomes.

Endosome-to-cytosol transport of viral nucleocapsids

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra- endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid ( LBPA) and its putative effector Alix/ AIP1, and is regulated by phosphatidylinositol- 3-phosphate ( PtdIns( 3) P) signalling via the PtdIns( 3) P- binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back- fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns( 3) P and their effectors.

Cholesterol and fatty acids regulate dynamic caveolin trafficking through the golgi complex and between the cell surface and lipid bodies

Caveolins are a crucial component of plasma membrane (PM) caveolae but have also been localized to intracellular compartments, including the Golgi complex and lipid bodies. Mutant caveolins associated with human disease show aberrant trafficking to the PM and Golgi accumulation. We now show that the Golgi pool of mainly newly synthesized protein is detergent-soluble and predominantly in a monomeric state, in contrast to the surface pool. Caveolin at the PM is not recognized by specific caveolin antibodies unless PM cholesterol is depleted. Exit from the Golgi complex of wild-type caveolin-1 or -3, but not vesicular stomatitis virus-G protein, is modulated by changing cellular cholesterol levels. In contrast, a muscular dystrophy-associated mutant of caveolin-3, Cav3P104L, showed increased accumulation in the Golgi complex upon cholesterol treatment. In addition, we demonstrate that in response to fatty acid treatment caveolin can follow a previously undescribed pathway from the PM to lipid bodies and can move from lipid bodies to the PM in response to removal of fatty acids. The results suggest that cholesterol is a rate-limiting component for caveolin trafficking. Changes in caveolin flux through the exocytic pathway can therefore be an indicator of cellular cholesterol and fatty acid levels.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3' leader-N-4a(P)-4b-M-G-L-5' trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus. (c) 2005 Elsevier B.V. All rights reserved.

A study in radiopaque polymeric materials

The problems associated with x-ray-transparent denture base are defined and conventional approaches to their solution are assessed. Consideration of elemental absorption parameters leads to the postulation that atoms such as zinc, and bromine, may be effective radiopacifiers over at least part of the clinical x-ray spectrum. These elements had hitherto been considered too light to be effective. Investigation of copolymers of methylmethacrylate and p-bromostyrene revealed no deleterious effects arising from the aromatically brominated monomer (aliphatic bromination caused UV destabilisation). For effective x-ray absorption a higher level of bromination would be necessary, but the expense of suitable compounds made further study unjustifiable. Incorporation of zinc atoms into the polymer was accomplished by copolymerisation of zinc acrylate with methylmethacrylate in solution. At high zinc levels this produced a powder copolymer convenient for addition to dental polymers in the dough moulding process. The resulting mouldings showed increasing brittleness at high loadings of copolymer. Fracture was shown to be through the powder particles rather than around them, indicating the source of weakness to be in the internal structure of the copolymer. The copolymer was expected to be cross-linked through divalent zinc ions and its insolubility and infusibility supported this. Cleavage of the ionic cross links with formic acid produced a zinc-free linear copolymer of high molecular weight. Addition of low concentrations of acrylic acid to the dough moulding monomer appeared to 'labilise' the cross links producing a more homogeneous moulding with adequate wet strength. Toxicologically the zinc-containing materials are satisfactory and though zinc is extracted at a measurable rate in an aqueous system, this is very small and should be acceptable over the life of a denture. In other respects the composite is quite satisfactory and though a marketable product is not claimed the system is considered worthy of further study.

Satisfação e impacto da saúde oral na qualidade de vida em pacientes reabilitados com prótese total dupla

Introduction: The rehabilitation of edentulous patients is one of the main challenges of Dentistry. Objective: To assess the satisfaction and the impact of oral health on quality of life of patients rehabilitated with conventional complete dentures in both arcades. Method: A clinical trial with 25 patients rehabilitated with CCD was conducted. The impact of oral health in individuals quality of life was evaluated through the Brazilian version of the OHIP-Edent and the patient satisfaction was evaluated using a specific questionnaire. The instruments were applied before and 3 months after rehabilitation. Results: There was a statistically significant improvement (p <0.001) on the impact of the quality of life of the denture users after the new rehabilitation. The analysis of 4 OHIP domains also showed statistically significant improvement (p <0.001). The final overall satisfaction with the dentures was statistically superior to initial satisfaction (p <0.001). The analysis of the specific aspects of satisfaction with CCD after 3 months of rehabilitation also showed significant results in the improvement of every aspect. Conclusion: Patients disappointed with their dentures, have their quality of life improved after the new rehabilitation, reducing the negative impact of the prostheses on oral health, and improving their satisfaction with the rehabilitation.

Effect of acetyl resin retentive arms on the retentive force ofcircumferential clasps: an in vitro study.

PURPOSE: To compare and to evaluate the stability of the retentive force of cobalt-chromium (Co-Cr) circumferential clasps (control) to those with an acetyl resin retentive arm. METHODS: Sixteen specimens with a couple of circumferential clasps were made using Co-Cr over a metal model providing 0.25 mm undercuts. Eight specimens were fabricated without the anterior retentive arm, which was made later using acetyl resin (Dental D). Insertion and removal simulation test was performed through 7250 cycles. The retentive force was recorded in Newtons (N) for periods corresponding to 0, 1, 2, 3, 4, and 5 years. The data were subjected to ANOVA and Tukey test to compare periods and to Student's t test to compare groups (α=0.05). RESULTS: Mean (SD) is presented for Co-Cr and resin groups, respectively: 8.09(3.05) and 2.79(1.57) in period 0; 10.48(4.25) and 3.32(1.92) in 1 year; 10.09(4.15) and 3.47(1.81) in 2 years; 9.87(4.30) and 3.46(1.87) in 3 years; 9.46(3.93) and 3.27(1.59) in 4 years; 9.63(3.79) and 3.41(1.59) in 5 years. There were significant differences for Co-Cr between periods of 0 and 1 (p<0.001), 0 and 2 (p<0.01) and 0 and 3 (p<0.05). In the resin group, no significant differences were found between periods (p>0.05). Comparisons between the groups showed statistical differences for all tested periods: 0 (p=0.0012), 1 (p=0.0013), 2 (p=0.0019), 3 (p=0.0031), 4 (p=0.0027) and 5 years (p=0.0014). CONCLUSIONS: Acetyl resin retentive arms, even if only in the anterior clasps, can significantly reduce the retentive force, but this force remained stable after 5 years of simulated use.

Does a mandibular RDP and new maxillary CD improve masticatory efficiency and quality of life in patients with a mandibular Kennedy class I arch?

Objectives To evaluate the change in masticatory efficiency and quality of life of patients treated with mandibular Kennedy class I removable partial dentures (RPDs) and maxillary complete dentures at the Department of Dentistry of the Federal University of Rio Grande do Norte. Materials and methods A total of 33 Kennedy class I patients were rehabilitated with maxillary complete dentures, and mandibular RPDs were selected for this non-randomized prospective intervention study. The patients had a mean age of 59.1 years. Masticatory efficiency was evaluated by colorimetric assay using fuchsin capsules. The measurements were conducted at baseline and 2 and 6 months after prosthesis insertion. Quality of life was evaluated using the Oral Health Impact Profile (OHIP-14) at baseline and 6 months after denture insertion. The Kolmogorov-Smirnov normality test was applied. Masticatory efficiency was evaluated by repeated measures ANOVA. Oral health-related quality of life was compared using the paired t test. Results There was no statistically significant difference in masticatory efficiency after denture insertion (p = 0.101). Significant differences were found (p = 0.010) for oral health-related quality of life. A significant improvement in psychological discomfort (p < 0.01) and psychological disability (p < 0.01) was observed. Mean difference value (95 % confidence interval) was 6.8 (3.8 to 9.7) points, reflecting a low impact of oral health on quality of life, considering the 0–56 range of variation of the OHIP-14 and a Cohen’s d of 1.13. Conclusion According to the results of the present study, rehabilitation with Kennedy class I RPDs and complete dentures did not influence masticatory efficiency but improved oral health-related quality of life. Clinical relevance The association between the patient’s quality of life and the masticatory efficiency is important for treatment predictability.

Duplicação rápida de prótese total: passo-a-passo

This study presents a simple, fast and low cost technique for fabrication new conventional dentures from the duplication of old prosthesis in use by the patient. Colorless acrylic resin was poured into the moulds obtained by duplication of prosthesis. With the replicas obtained a functional impressions using polyether should be performed and they are stabilized with occlusal registration in acrylic resin. The molds need to be castings and mounted on an semi-adjustable articulator. The artificial teeth are positioned with the assistance of a guide made condensation silicone to reproduce the positioning of the teeth of the old prosthesis and fixed with wax 7. After approval of the teeth on the trial in wax, without adjustment of the planes, the prosthesis may be processed in the laboratory. After occlusal adjustment in the articulator the same can be installed.