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Abstract The construction of expression vectors derived from the human adenovirus type 5 (Ad5), usually based on homologous recombination, is time consuming as a shuttle plasmid has to be selected before recombination with the viral genome. Here, we describe a method allowing direct cloning of a transgene in the E3 region of the Ad5 genome already containing the immediate early CMV promoter upstream of three unique restriction sites. This allowed the construction of recombinant adenoviral genomes in just one step, reducing considerably the time of selection and, of course, production of the corresponding vectors. Using this vector, we produced recombinant adenoviruses, each giving high-level expression of the transgene in the transduced cells.

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.

High-risk human papillomavirus (HPV) types 16 and 18 are associated with more than 70% of cervical cancer cases. The oncoprotein E6 is multifunctional and has numerous cellular partners. The best-known activity of E6 is the polyubiquination of the pro-apoptotic tumor suppressor p53, targeting it for degradation by the 26S proteasome. Loss of p53 triggers genomic instability and favors cancer development. Here, we generated recombinant adenovirus (Ad) vectors expressing artificial microRNAs directed against HPV16 E6 (Ad16_1) or HPV18 E6 (Ad18_2). E6-knockdown was observed in HeLa after treatment with Ad18_2 and in SiHa with Ad16_1. Western-blot experiments found an increase in p53 levels after treatment in both cell lines. Cell death was observed in both cell lines after knockdown of E6. Further analysis such as cleavage of caspases (3 and 7) as well as of PARP1 indicated that treated HeLa and SiHa cells underwent apoptosis. The growth of HeLa-derived tumors developed in nude mice was significantly reduced after intra-tumoral injection of Ad18_2. Therefore, vectorisation of artificial miRNA against E6 oncoprotein by means of recombinant adenoviruses might represent a valuable therapeutic approach for treating HPV-positive cancers., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.

The E6 oncoprotein produced by high-risk mucosal HPV stimulates ubiquitinylation and proteasome-dependent degradation of the tumour suppressor p53 via formation of a trimeric complex comprising E6, p53, and E6-AP. p53 is also degraded by its main cellular regulator MDM2. The main binding site of p53 to MDM2 is situated in the natively unfolded N-terminal region of p53. By contrast, the regions of p53 implicated in the degradation by viral E6 are not fully identified to date. Here we generated a series of mutations (Y103G, Y107G, T155A, T155V, T155D, L264A, L265A) targeting the central folded core domain of p53 within a region opposite to its DNA-binding site. We analysed by in vitro and in vivo assays the impact of these mutations on p53 degradation mediated by viral E6 oncoprotein. Whereas all mutants remained susceptible to MDM2-mediated degradation, several of them (Y103G, Y107G, T155D, L265A) became resistant to E6-mediated degradation, confirming previous works that pointed to the core domain as an essential region for the degradation of p53. In parallel, we systematically checked the impact of the mutations on the transactivation activity of p53 as well as on the conformation of p53, analysed by Nuclear Magnetic Resonance (NMR), circular dichroism (CD), and antibody probing. These measurements suggested that the conformational integrity of the core domain is an essential parameter for the degradation of p53 by E6, while it is not essential for the degradation of p53 by MDM2. Thus, the intracellular stability of a protein may or may not rely on its biophysical stability depending on the degradation pathway taken into consideration.

The construction of expression vectors derived from the human adenovirus type 5 (Ad5), usually based on homologous recombination, is time consuming as a shuttle plasmid has to be selected before recombination with the viral genome. Here, we describe a method allowing direct cloning of a transgene in the E3 region of the Ad5 genome already containing the immediate early CMV promoter upstream of three unique restriction sites. This allowed the construction of recombinant adenoviral genomes in just one step, reducing considerably the time of selection and, of course, production of the corresponding vectors. Using this vector, we produced recombinant adenoviruses, each giving high-level expression of the transgene in the transduced cells.

Human group C adenoviruses can infect many cell types, and this is due to the widespread expression of their receptor, the coxsackievirus and adenovirus receptor (CAR). Adenovirus vectors for cancer gene therapy could be significantly improved if their tropism were restricted to tumor cells. In this work, previously identified peptides that target human papillomaviruses (HPV)-transformed cells were inserted into the HI loop of a non-CAR-binding fiber. These modified fiber proteins were able to assemble into adenovirus particles. We demonstrated that these modifications ablated the native tropism of adenovirus type 5, and these modified adenoviruses were shown to preferentially transduce HPV-transformed cell lines.

The E6 protein of human papillomavirus type 16 (16E6) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We have designed a strategy for neutralizing 16E6 based on the intracellular expression of single-chain Fv antibodies (scFvs) specific to 16E6. Recombinant adenovirus vectors were constructed to allow expression of two 16E6-binding scFvs and one 16E6-non-binding scFv in HPV16-positive and -negative cells. Expression of the scFvs provoked two types of effects: (i) inhibition of proliferation of all cell lines tested, this aspecific toxicity being likely due to the aggregation of unfolded scFvs; and (ii) apoptosis observed only in HPV16-positive cervical cancer cell lines after expression of 16E6-binding scFvs, this specific effect being proportional to the intracellular solubility of the scFvs. These data demonstrate the feasibility of intracellular immunization with anti-16E6 scFvs and highlight the importance of the solubility of the intracellular antibodies.

The E6 protein of cancer-associated human papillomavirus type 16 (16E6) binds to p53 and, in association with E6AP, promotes its degradation through the ubiquitin-proteasome pathway. The aim of this work was to develop monoclonal antibodies against 16E6 and to test their effect on the binding of 16E6 to p53 and E6AP, and on the degradation of p53. It was shown that an antibody directed against the N terminus of 16E6 inhibited E6AP-dependent binding to p53 and degradation of p53, whereas two different antibodies directed to the second zinc-binding domain of 16E6 reduced 16E6 E6AP-independent binding to p53 and binding to E6AP but not degradation of p53.

The early transcription unit 3 (E3) of adenoviruses (Ads) encodes immunomodulatory functions. We previously described a novel gene of 49K within the E3 region of Ad19a, an Ad of subgenus D that is similar to Ad8 and Ad37 causes epidemic keratoconjunctivitis (EKC). Interestingly, 49K was reported not to be present in Ad9 and Ad17, other subgenus D Ads not causing EKC. Therefore, we investigated whether 49K is selectively expressed in EKC-causing Ads. Using specific DNA probes, we detect 49K-homologous genes in all subgenus D Ads tested. Moreover, 49K-specific antibodies recognize a high molecular weight protein in cells infected with all subgenus D serotypes irrespective of their ability to cause EKC. Sequencing of several 49K genes reveals a high homology without a distinct feature recognizable for those of EKC-associated Ad strains. Thus, E3/49K is a subgenus D specific E3 protein whose expression does not correlate with the EKC-causing phenotype and thus may rather be implicated in illnesses commonly caused by this subgenus. Interestingly, the 49K sequences of Ad19a and Ad37 are identical. To estimate the extent of the sequence identity between these two viruses, we initially sequenced the right ITR and the hexon. This analysis revealed that the right ITR of Ad19a is identical to Ad37, while the hexon sequence is Ad19p-like. This suggested that the region of identity is much larger and that Ad19a arose by recombination of Ad37 with an Ad19p-like Ad. Further sequencing mapped the crossover within the DNA binding protein. Thus, Ad19a contains a large sequence block ( approximately 13 kb), from the 100K gene to the right ITR, identical to Ad37. The implications of these findings in light of the temporal appearance of the EKC-causing Ad strains are discussed.

This article describes the possibilities and difficulties of reconstruction in the front regio by means of implants and bone transplants in five patients. First of all, planning is the most important step. Sometimes the class III relationship is a real challenge. Different bone transplants, the inlay and onlay technique, the number of the implants, the importance of the way the implants are uncovered in the second stage are discussed. In the front region, we aim for functional and esthetic reconstruction.

The presentation of a case where an agenetic lateral tooth was replaced by an oral implant, has been described. The following parameters are important: oral hygiene, the moment of implant surgery, presurgical orthodontic treatment, alternative prosthetic treatment possibilities and recall of the patient.

This article describes the treatment of a boy with Class II deep bite and hypoplasia of the alveolar process due to agenesis of the canines and premolars. At first, the occlusion has been adjusted by advancement of the mandible and opening of the bite after bilateral sagittal split osteotomy. Later, under local anaesthesia, a segmental osteotomy of the hypoplastic alveolar process has been performed. Distraction of the hypoplastic alveolar process has been achieved by orthodontic traction on the residual dentition in the segments. After sufficient augmentation of the alveolar process, 6 implants for two bridges have been placed.

Preliminary analysis of Atlantic salmon alpha- and beta-globin genes indicated that these genes are linked in a 3' to 3' orientation, with the RNA-coding sequences located on opposite strands. In this report, we show that two different alpha-globin genes have the same orientation and are encoded on the same strand whereas two different beta-globin genes are encoded on the opposite strand and also have the same orientation. This cluster of globin genes is divided into two subclusters: one for the Bohr globin genes and one for the non-Bohr globin genes. This is the first evidence for this type of arrangement found for globin genes. DNase I footprint analysis of two of the globin promoters show erythroid-specific transcription factor binding sites that have also been found in human and other mammalian globin genes.

Human adenovirus can cause persistent infections in man. Implicated in this phenomenon is the early transcription unit 3 (E3) of the virus which encodes proteins that are primarily devoted to counteract the lytic attack by the host immune system: Several E3 proteins (14.7K, 10.4K and 14.5K) protect infected cells from the lytic activity of tumor necrosis factor alpha (TNF) while the most abundant E3 protein, E3/19K, inhibits lysis by cytotoxic T cells. E3/19K interacts with class I histocompatibility (MHC) antigens in the rough endoplasmic reticulum, thereby preventing transport of MHC molecules to the cell surface and, consequently, MHC-restricted T cell recognition. In addition, the 10.4K and 14.5K proteins downregulate cell surface expression of the epidermal growth factor receptor. Interestingly, adenovirus-mediated pneumonia in mice is accompanied by induction of TNF, a cytokine known to enhance MHC expression. We previously showed that TNF is unable to restore MHC class I expression in E3/19K transfected cells but rather leads to a further reduction of MHC antigens. This effect correlated with an increased production of E3/19K mRNA and protein. We now find in addition an upregulation of other E3 proteins in transfected as well as in infected cells. This coordinated upregulation of E3 proteins indicates that TNF stimulates the E3 promoter, probably by activating the transcription factor NF-kappa B. Thus, a novel interaction between the immune system and adenovirus is described in which the virus takes advantage of an immune mediator to promote expression of several immunosubversive proteins supporting its escape from immunosurveillance.

The sequence of the C-terminal 1058 amino acids of atlantic salmon (Salmo salar) apolipoprotein (apo) B was deduced from the nucleotide sequence of cloned cDNA. In comparison with chicken or mammals apoB-100, salmon apoB is C-terminally truncated and extended gaps are found. The two clusters of positively charged residues, previously identified as part of the putative low-density-lipoprotein (LDL) receptor-binding domain of apoB, are brought into close proximity in salmon apoB. This is achieved by the absence between the two clusters of the proline-rich area with the potential to form an amphipathic beta sheet, present in higher vertebrates. In addition, analysis of apoB amino acid sequences currently available in vertebrates revealed the presence of an extended internal duplication in the putative LDL receptor-binding domain. Thus, the two basic clusters would have been duplicated resulting in the presence, except for salmon apoB, of two homologous sites in the C-terminal part of the molecule. The results described here together with earlier biochemical and genetic evidence support the view that Arg3500, a residue mutated in familial defective apoB-100, could be included in a folded critical region of the putative LDL receptor-binding domain of human apoB-100. This region possibly brings the two sub-domains that arise from the duplication close to each other.

cDNA clones coding for the transcription factor HNF1 have been isolated from Atlantic salmon (Salmo salar L.). The 559 amino acid residue long encoded protein shows high conservation, with respect to other species, of the domains necessary for DNA-binding: the HNF1 atypical homeodomain, the POU related sequence and the dimerisation domain. Alignment with rat HNF1 protein reveals that the transcription activation domains ADI and ADIII are relatively conserved in the fish sequence whereas ADII is not. Phylogenetic analysis indicates that higher vertebrate HNF1s and the related variant HNF1s (vHNF1s) are more closely related to each other than any of them is to Salmon HNF1, suggesting that the duplication event from which HNF1 and vHNF1 genes arose occurred after the divergence of the tetrapod and teleost ancestors. Northern blot analysis show a single transcript, of about 2.6 kb, which is not exclusive to liver but is also present in intestine, kidney and spleen. Using polymerase chain reaction (PCR) we have isolated the salmon albumin gene promoter which contains, upstream of the TATA box, a potential binding site for HNF1. The salmon HNF1 protein synthesized by in vitro transcription-translation of the full-length cDNA is able to bind specifically with equivalent affinities to either the rat or salmon albumin promoter.

The authors give a review of the literature on the different onlay and inlay bone graft techniques in reconstruction of the extremely resorbed maxilla. Own experiences and results of onlay and inlay bone grafting in combination with implants are described. A treatment protocol used since September 1989 and the outcome of 198 implants placed in combination with different reconstruction techniques are presented.

A radiological technique, using a new CT software program for the evaluation of alveolar process height and width, is presented. Irradiation is kept within acceptable limits when this technique is used. Measurements obtained with this technique were compared with those obtained on panoramic radiographs in 40 "half-jaws" (21 maxillar and 19 mandibular). We found that new indications for implantation emerge in the mandibular region because 'Denta Scan' can sometimes show possibilities to place implants on the buccal side of the canal (2 of 19 mandibular cases) when no possibilities are present above the canal on both the panoramic radiographs and Denta Scan images. In the maxillar region Denta Scan avoids unnecessary interventions by demonstrating the insufficient width of the alveolar ridge, often missed on panoramic radiographs (4 of 21 maxillar cases). Moreover the use of Denta Scan allows the use of implants with optimal length and diameter (23 of the 40 cases), giving better long-term results.