Computer-guided gap arthroplasty: a new approach to the execution of preplanned osteotomies for the treatment of bony ankylosis of the temporomandibular joint.

The purpose of this study was to assess the efficacy of 3-dimensional, printed, patient-specific guides to direct virtual gap arthroplasties that were designed for five patients with advanced unilateral ankylosis of the temporomandibular joint. The guides were used to mimic the intraoperative creation of five preplanned osteotomies, as well as simulating the width and depth of the bone cleavage. The accuracy of the devices in guiding the surgical simulation was assessed by superimposing the preoperative and postoperative computed tomographic scans. The devices were easily put in place with smooth uniform surgical bone cleavage, and favourable postoperative outcomes. The statistical analysis between the planned and surgical gaps, showed that the difference in dimensions was not significant (p=0.1018). The patient-specific gap arthroplasty was neither too near the skull base nor did it jeopardise the height of the mandibular ramus.

Assessment of vertical ridge augmentation in anterior aesthetic zone using onlay xenografts with titanium mesh versus the inlay bone grafting technique: A randomized clinical trial.

The aim of this study was to evaluate the final vertical gain at the deficient anterior maxillary alveolar ridges using onlay bone grafts with titanium mesh versus inlay bone grafting. This was a single institutional randomized comparative clinical trial. The study population included 16 patients, with edentulous anterior maxillary alveolar ridges (40 implant sites) who were presented and treated at the Faculty of Oral and Dental Medicine in Cairo University from September 2013 to August 2015. Selected patients were randomly divided into two equal groups. The control group received onlay particulate xenograft together with titanium mesh as a space-maintaining device while the study group received inlay block xenograft (sandwich osteotomy) fixed with mini-plates. Assessment using cone beam computed tomography (CBCT) included the mean percentage of vertical gain at the proposed implant sites after 6 months taken from cross-sectional cuts. A total of 40 delayed implant placements were done. Results showed that there was no statistical significance between the two groups (P=0.2); the mean percentage of 6 months postoperative vertical bone gain in the control group was 20.7% and that in the study group was 31.6%.

ANTIMICROBIAL ACTIVITY FOR EXCRETION AND SECRETION OF THE GREENBOTTLE FLY LARVAE LUCILIA SERICATA (MEIGEN) (DIPTERA: CALLIPHORIDAE).

Sterile larval excretion/secretion (ES) exhibited antibacterial activity against some species of bacteria. They were shown to inhibit the growth of Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae and Fungi Geotricum candidum and Aspergillus fumigatus thus exhibited limited inhibitory effect towards Gram-positive bacteria Streptococcus pyogenes and Staphylococcus epidermidis and Gram-negative Proteous vulgaris and Fungi Syncephalastrum racemosum, Candida albicans, that effect was slowed down when challenged with secretion on a solid media but no zone of complete inhibition was detectqd. Growth inhibiting activity was determined in liquid growth media using the Gram-positive, Gram-negative bacterial and fungal strains as indicator organisms.

Oral and subcutaneous absorption of insulin poly(isobutylcyanoacrylate) nanoparticles.

Dispersions of insulin poly(isobutylcyanoacrylate) nanoparticles were obtained by anionic in situ polymerization using aqueous pluronic acid solution. Results showed a decrease in particle size diameter by increasing the pluronic acid concentration. Nanoparticles prepared in the presence of 2.5% pluronic acid resulted in particles of 85 nm average diameter and 59% intra-particular insulin load without the use of the oily core [Damge, C., Michel, M., Aprahamian, M., Couveur, P., 1988. New approach for oral administration with polycyanoacrylate nanocapsules as drug carrier. Diabetes 37, 246-251]. In vivo testing was performed on streptozocin induced diabetic rats. The subcutaneous injection of insulin nanoparticles was able to prolong its duration of hypoglycemic effect from 6 to 72 h. Effective oral absorption of the entrapped insulin was significantly better (p<0.01) when compared with non-encapsulated insulin or the control experiments.

Oral absorption of insulin encapsulated in artificial chyles of bile salts, palmitic acid and alpha-tocopherol dispersions.

The hypoglycemic effect of orally given insulin was studied on rabbits, using different bile salts as absorption promoters, in two different carriers to form an artificial chyloform system ready to be absorbed by intestinal mucosa. The rank order of enhancement by bile salts in the presence of 1% ethanol was deoxycholate>cholate>glycocholate>glycodeoxycholate>taurodeoxycholate>no bile salts. The dose response studies with increased insulin loaded in the chyle showed a greater corresponding hypoglycemic effect with the system of cholate-palmitic-alpha-tocopherol dispersions than the cholate-palmitic acid dispersions. A more effective hypoglycemic effect was achieved using lower doses of the deoxycholate-palmitate-tocopherol-chyle dispersions.

Parallel solid-phase synthesis of nucleoside phosphoramidate libraries.

Combinatorial chemistry is playing an increasingly prominent role in the process of drug discovery. A nucleic acid-based (NAB) scaffold can be engineered to create functional group and topological diversity in a library. Described herein is the parallel solid-phase synthesis of combinatorial libraries of nucleoside phosphoramidates, and the first evaluation of antiviral activity against hepatitis B virus (HBV).

Inhibition of tumor growth with doxorubicin in a new orthotopically implanted human hepatocellular carcinoma model.

A number of human xenograft orthotopic models of hepatocellular carcinoma (HCC) have been previously established by growing histologically-intact patient specimens in nude mice. However, the availability of HBV and HCV negative human hepatocellular carcinoma specimens is scarce and the pattern of tumor growth in nude mice varies depending on the tumor type. In the present study, we have established a reproducible xenograft orthotopic model using a human HCC cell line designated HuT7-3 that was derived from two rounds of subcloning of the parental Huh-7 cell line. The tumor growth rate of the HuT7-3 cell line, grown at a primary subcutaneous site, was markedly higher than that of the Huh-7 parental cell line or the human hepatoblastoma Hep-G2 cell line. Furthermore, we have shown that doxorubicin, when administered intravenously, is efficient in inhibiting the development of subcutaneous tumor but leads to the regression of the orthotopic human HCC. Consequently, this novel HCC xenograft orthotopic model can be used for the evaluation of antitumor drugs.

Hepatitis C viral IRES inhibition by phenazine and phenazine-like molecules.

An in vitro assay based on the expression of Fluci reporter gene under the translational control of HCV IRES was used to evaluate and screen compound libraries. A structure-activity relationship study on a phenazine hit was conducted. Our data suggest that an intact phenazine or phenazine-like core with two distal polar substitutions is crucial for potency.

The hepatitis C virus NS5B RNA-dependent RNA polymerase activity and susceptibility to inhibitors is modulated by metal cations.

OBJECTIVES: The aim of this study was to understand the effect of metal cations on the hepatitis C virus (HCV) NS5B in vitro RNA-dependent RNA polymerase (RdRp) activity and its susceptibility to various inhibitors. METHODS: A recombinant full-length HCV NS5B protein was expressed in insect cells and purified to homogeneity. RdRp activity was assessed using standard filtration or polyacrylamide gel-based assays. RESULTS: Efficient inhibition of the HCV NS5B RdRp activity by gliotoxin, as well as by various substrate analogs, occurs in the presence of Mn2+, but not of Mg2+. Assays performed in the presence of both cofactors suggest that, in vitro, the enzyme's affinity for Mn2+ is higher than that for Mg2+. In addition, the RdRp activity, displayed in the presence of heteropolymeric templates, is significantly increased when the metal cofactor consists of Mn2+. Finally, steady state kinetics showed that the velocity of the reaction, as well as the affinity of the enzyme for its substrate, could both be affected by the nature of the divalent metal cation used.

Survival and proliferation of cells expressing caspase-uncleavable Poly(ADP-ribose) polymerase in response to death-inducing DNA damage by an alkylating agent.

To determine whether caspase-3-induced cleavage of poly(ADP-ribose) polymerase (PARP), a DNA damage-sensitive enzyme, alters the balance between survival and death of the cells following DNA damage, we created stable cell lines that express either caspase-uncleavable mutant or wild type PARP in the background of PARP (-/-) fibroblasts. The survival and apoptotic responses of these cells were compared after exposure to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a DNA-damaging agent that activates PARP, or to tumor necrosis factor-alpha, which causes apoptosis without initial DNA damage. In response to MNNG, the cells with caspase-uncleavable PARP were very resistant to loss of viability or induction of apoptosis. Most significantly, approximately 25% of these cells survived and retained clonogenicity at a level of DNA damage that eliminated the cells with wild type PARP or PARP (-/-) cells. Expression of caspase-uncleavable PARP could not protect the cells from death induced by tumor necrosis factor, although there was a slower progression of apoptotic events in these cells. Therefore, one of the functions for cleavage of PARP during apoptosis induced by alkylating agents is to prevent survival of the extensively damaged cells.

Kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus.

The kinetics of appearance of antibodies directed to the major structural proteins N, M and E of porcine reproductive and respiratory syndrome virus (PRRSV) was followed in pigs naturally- and experimentally-exposed to the virus. Specific IgM antibody titers were first detected by indirect immunofluorescence (IIF) at the end of the first week of PRRSV infection, peaked by day 14 to 21 post-inoculation (p.i.), then rapidly decreased to undetectable levels by day 35 to 42 p.i. On the other hand, specific IgG antibody titers peaked by day 21 to 28 p.i. and remained unchanged to the end of the 6- or 9-week observation period; in addition, a persistent viremia was observed. Virus neutralizing (VN) antibody titers > 8 were not detected until 3 to 4 weeks p.i. Taken together, the results obtained by Western blotting analyses using purified virus and E. coli-expressed ORFs 5 to 7 gene products, suggested that antibodies directed against the envelope E protein appear by day 7 p.i., whereas antibodies directed against the nucleocapsid N and membrane M proteins can only be detected by the end of the second week p.i. No correlation could be demonstrated between VN and IIF antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i.

Sequence and expression of the ns2 protein gene of human coronavirus OC43.

The complete nucleotide sequence of the ns2 gene of human coronavirus OC43 (HCV-OC43) was determined. Sequence analysis revealed an open reading frame that could encode a protein of 278 amino acids, with an estimated molecular mass of 32.2 kDa. Six potential phosphorylation sites are present but no sites of N-glycosylation were found. The amino acid sequence of the HCV-OC43 ns2 protein shows 92% identity with that of the Mebus strain of bovine coronavirus (BCV). However, a stretch of nine consecutive amino acids near the C terminus is completely different, causing it to be very hydrophilic, which contrasts with the hydrophobic nature of this region in BCV. As shown by immunofluorescence with a monospecific antiserum, the ns2 protein was expressed in the cytoplasm of HCV-OC43-infected HRT-18 cells.

Structural gene analysis of a Quebec reference strain or porcine reproductive and respiratory syndrome virus (PRRSV).

The 3' end genomic region of a Québec PRRSV reference strain (IAF-exp91), propagated in porcine alveolar macrophages (PAM), was sequenced and compared to the prototype European strain, the Lelystad virus (LV). The sequence, which represents the 3'-terminal 2834 nucleotides, encompassed 5 ORFs corresponding to ORFs 3 to 7 of LV. Extensive genomic variations resulting from an important rate of nucleotide additions, substitutions, and deletions were demonstrated between the two viruses. Indeed, the two corresponding sequences displayed a total of 66% and 63% identity at the nucleotide and amino acid levels, respectively. The predicted products of ORFs 5, 3, and 7, showed the highest rate of amino acid variations with percentages of identity of 52, 54, and 59, respectively. Sequence analysis of an additional Québec strain that could be propagated in a continuous cell line (MARC-145), suggested that Québec PRRSV strains belong to a genotype distinct from that of LV, thus confirming previous serological results which allowed to divide PRRSV isolates into two distinct antigenic subgroups (U.S. and European). Six viral major polypeptides with apparent M(rs) of 14.5K, 15K, 19K, 24.5K, 29K, and 42K could be identified from lysates of viral infected cells, of which the 15, 19 and 24.5K species seemed to be structural. In vitro translation products of ORFs 7 and 6 comigrated with the 15 and 19 K viral proteins, whereas that of ORF 5 may be associated to the 24.5K when translated in presence of microsomes. Consequently, it is likely that ORFs 7 to 5, encode the three major structural proteins.

Identification and characterization of the porcine reproductive and respiratory virus ORFs 7, 5 and 4 products.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes abortions and respiratory diseases in pigs. The PRRSV genome is a positive-sense polyadenylated RNA molecule of about 15 kb. Along with the genes for structural proteins (envelope, matrix and nucleocapsid proteins) PRRSV genome contains a number of ORFs potentially encoding nonstructural proteins (ns). To investigate the nature of the PRRSV ORFs 7, 5 and 4 products, we have cloned the envelope (ORF5) nucleocapsid (ORF7) and ns4 (ORF4) protein genes in the bacterial expression vector pMAL(TM)-c2 under control of the "tac" promoter and expressed the proteins in E. coli. The recombinant proteins were recognized by porcine and rabbit hyperimmune serums to PRRSV, suggesting their structural nature.

Molecular analysis of the ORFs 3 to 7 of porcine reproductive and respiratory syndrome virus, Québec reference strain.

The cDNA sequence of the 3'-terminal genomic region of the Québec IAF-exp91 strain of porcine reproductive and respiratory syndrome virus (PRRSV) was determined and compared to those of other reference strains from Europe (Lelystad virus) and US (ATCC VR2385, MN-1b). The sequence (2834 nucleotides) which encompassed ORFs 3 to 7 revealed extensive genomic variations between the Québec strain and Lelystad virus (LV), resulting from high number of base substitutions, additions and deletions. The ORFs 5, 3, and 7 seemed to be relatively the most variable; the predicted encoding products of the Québec and LV strains displayed only 52%, 54%, and 59% amino acid identities, respectively. Nevertheless, in vitro translation experiments of the structural genes (ORFs 5, 6, and 7) and radioimmunoprecipitation assays with extracellular virions gave results similar to those previously reported for LV. In contrast, close genomic relationships were demonstrated between Québec and US strains. Taking together, these results indicate that, although structurally similar, North American PRRSV strains belong to a genotype distinct from that of the LV, thus supporting previous findings that allowed to divide PRRSV isolates into two antigenic subgroups (U.S. and European).

Detection of porcine reproductive and respiratory syndrome virus and efficient differentiation between Canadian and European strains by reverse transcription and PCR amplification.

Two sets of oligonucleotide primers (1008PS-1009PR and 1010PLS-1011PLR) were designed according to the sequence of the nucleocapsid protein (N) gene of Quebec reference strain IAF-exp91 of porcine reproductive and respiratory syndrome virus (PRRSV). The primers were used in reverse transcription and PCR (RT-PCR) experiments for detection of viral genomic RNA either from infected porcine alveolar macrophages (PAM) or tissues from experimentally infected specific-pathogen-free pigs. Considering the high degree of variation detected between the nucleotide sequences of the N genes of IAF-exp91 and Lelystad virus (LV) strains of PRRSV, the primers 1008PS-1009PR were referred to as the specific primers, since they were chosen in such a manner that they could amplify only sequences from IAF-exp91 RNA and not from LV. On the other hand, the primer pair 1010PLS-1011PLR was common to both strains of PRRSV. When analyzed by agarose gel electrophoresis, the products of RT-PCR from each set of primers were resolved as single band of the predicted size, the specificity of amplified products being confirmed by Southern blotting with a specific IAF-exp91 N gene probe. No amplification was observed when RNA was extracted from uninfected PAM or from other porcine viruses. As expected, only the common primer pair was able to amplify RNA from the Quebec reference strain and two European strains (LV and Weybridge). The resulting bands displayed differences in electrophoretic mobilities due to the absence of 37 nucleotides in both European strains, thus allowing their differentiation from the IAF-exp91 strain. Most of the tissue culture-adapted Quebec isolates were detected with both primer pairs. The sensitivity of the enzymatic amplification method for detection of PRRSV from lung tissues was a 50% tissue culture infective dose of 5. RT-PCR was found to be more sensitive than indirect immunofluorescence assay for detection of PRRSV in tissues from experimentally infected pigs and as sensitive as virus isolation in PAM, especially when combined with Southern blotting with the digoxigenin-labeled N probe and chemiluminescence detection.

Identification of major differences in the nucleocapsid protein genes of a Québec strain and European strains of porcine reproductive and respiratory syndrome virus.

The sequence of the 3'-terminal region of the genome of Québec reference strain IAF-exp91 of porcine reproductive and respiratory syndrome virus (PRRSV) was investigated by analysis of four cDNA clones. The 3'-terminal 530 nucleotides (nt) encompassed a large open reading frame with a coding capacity of 123 amino acids (M(r) 13,649). The predicted protein was extremely basic and hence was considered to correspond to the nucleocapsid (N) protein gene. When compared to the homologous sequences of two reference Netherlands strains (Lelystad and isolate 10) of PRRSV, the IAF-exp91 N protein was found to be five amino acids shorter and displayed a high degree of divergence. Overall, IAF-exp91 strain showed identities of 63% and 59% with both reference European strains at the nucleotide and amino acid level, respectively. Two amino acid stretches, STAPM and SQGAS, present respectively at the N- and C-terminal regions of the N protein of European strains, were missing in the IAF-exp91 N protein sequence. The 3'-terminal non-coding region (151 nt) of the IAF-exp91 strain was 22 nt longer than that of the European strains. The aligned nucleotide sequence of this non-coding region exhibited an overall identity of 59% with that of the European strains. The Québec reference strain of PRRSV appeared to be related more closely to equine arteritis virus and lactate dehydrogenase-elevating virus than are the two European strains of the virus. Preliminary data obtained by reverse transcription-PCR experiments, using specific or common oligonucleotide primers, suggested that this approach could be useful for distinguishing between PRRSV strains from different geographic origins.

Porcine reproductive and respiratory syndrome virus: morphological, biochemical and serological characteristics of Quebec isolates associated with acute and chronic outbreaks of porcine reproductive and respiratory syndrome.

Cytolytic and noncytolytic strains of the porcine reproductive and respiratory syndrome virus (PRRSV) were isolated in primary cultures of porcine alveolar macrophages (PAM) from lung homogenates of stillborn fetuses or blood samples of dyspneic piglets collected from Quebec pig farms having experienced acute or chronic outbreaks of PRRS. Serological identification of the virus was confirmed by indirect immunofluorescence and indirect protein A-gold immunoelectron microscopy using reference antiserum prepared from experimentally-infected specific pathogen free (SPF) piglets and monoclonal antibodies (MoAbs) directed against the p15 nucleocapsid (N) protein of the reference ATCC-VR2332 isolate. Intracytoplasmic enveloped viral particles that tended to accumulate into cytoplasmic vesicles were observed in the infected PAM; no budding was demonstrated at the level of the cytoplasmic membrane. The extracellular virions appeared as pleomorphic but mostly spherical enveloped particles, 50-72 nm in diameter (averaged diameter of 50 particles was 58.3 nm), with an isometric core about 25-30 nm. Buoyant density of the virus in CsCL density gradients was estimated to 1.18-1.20 g/mL. No hemagglutinating activity was demonstrated. Analysis of semipurified virions of isolate IAF-exp91 by radioimmunoprecipitation (RIPA) and Western immunoblotting experiments, using reference rabbit and porcine hyperimmune sera, revealed four major viral proteins, a predominant 15 kD N protein and three other proteins with predicted M(r_ of 19, 26 and 42 kD. Progeny viral particles produced in PRRSV-infected PAM in the presence of tunicamycin lacked the 42 kD protein, thus confirming its N-glycosylated nature. Immunoprecipitation experiments using the anti-ATCC-VR2332 MoAbs confirmed the close antigenic relationships between Quebec and American reference isolates of PRRSV.

Molecular characterization of the S protein gene of human coronavirus OC43.

The gene encoding the spike protein of the OC43 strain of human coronavirus (HCV-OC43) was cloned and sequenced. The complete nucleotide sequence revealed an open reading frame of 4062 nucleotides encoding a protein of 1353 amino acids with a predicted M(r) of 150,078. Structural features include 22 N-glycosylation sites, an N-terminal hydrophobic signal sequence of 17 amino acids, an hydrophilic cysteine-rich sequence of 35 amino acids near the C terminus, and a potential proteolytic cleavage site (RRSR) between amino acid residues 758 and 759, yielding S1 and S2 segments of 84,730 and 65,366 M(r), respectively. The predicted amino acid sequence of the spike protein of HCV-OC43 has 91% identity with that of the Mebus strain of bovine coronavirus, revealing more sequence divergence in the putative bulbous part (S1) than in the predicted stem region (S2).