Optimization of a medium composition for the heterologous production of Alcaligenes faecalis penicillin G acylase in Bacillus megaterium.

Penicillin G acylase (PGA) is a strategic enzyme in the production processes of beta-lactam antibiotics. High demand for ß-lactam semisynthetic antibiotics explain the genetic and biochemical engineering strategies devoted towards novel ways for PGA production and application. This work presents a fermentation process for the heterologous production of PGA from Alcaligenes faecalis in Bacillus megaterium with optimization. The thermal stability from A. faecalis PGA is considerably higher than other described PGA and the recombinant enzyme is secreted to the culture medium by B. megaterium, which facilitates the separation and purification steps. Media optimization using fractional factorial design experiments was used to identify factors related to PGA activity detection in supernatant and cell lysates. The optimized medium resulted in almost 6-fold increased activity in the supernatant samples when compared with the basal medium. Maximum enzyme activity in optimized medium composition achieves values between 135 and 140 IU/ml. The results suggest a promising model for recombinant production of PGA in B. megaterium with possible extracellular expression of the active enzyme.

Tephrosia toxicaria (Sw.) Pers. extracts: Screening by examining aedicidal action under laboratory and field conditions along with its antioxidant, antileishmanial, and antimicrobial activities.

An increase in the incidence of arboviral, microbial and parasitic infections, and to disorders related to oxidative stress has encouraged the development of adjuvant therapies based on natural formulations, such as those involving plant extracts. Thus, to expand the repertoire of the available therapeutic options, this study aimed to describe the versatility of Tephrosia toxicaria (Sw.) (Pers., 1807) extracts for the control of arbovirus vectors, as well as their antioxidant, antileishmanial, and antimicrobial potential. Among the aqueous and hydroethanolic extracts obtained, the hydroethanolic extract from roots (RHA) was identified as the most active larvicide extract demonstrating, respectively, the lowest lethal concentration (mg/mL) for 50%, 90% and 99% of Aedes aegypti (L., 1762) and Aedes albopictus (S., 1894) larvae, observed at 24 h (0.33, 0.84 and 1.80; 0.32, 0.70 and 1.32) and 48 h (0.17, 0.51 and 1.22; 0.26, 0.47 and 0.78) post-exposure. Field assays revealed that RHA (0.84 mg/mL) is a potential oviposition deterrent, reducing egg-laying by approximately 90%. RHA (0.1 mg/mL) also exhibited antioxidant activity for the following tests: total antioxidant capacity (286.86 mg AAE/g), iron (87.16%) and copper (25.64%) chelation, and superoxide scavenging (10%). In the cell culture assays, RHA (0.1 mg/mL) promoted regeneration of metabolic activity (92% cell viability) in cells exposed to oxidative stress. Furthermore, RHA displayed weak antileishmanial activity (IC50 = 3.53 mg/mL) against Leishmania amazonensis and not exhibit antimicrobial activity. The extraction favored the concentration of carbohydrates in RHA, in addition to lectins and protease inhibitors, with molecular masses estimated between 10 and 24 kDa. Cytotoxicity and phytotoxicity analyses of RHA suggested its biosecurity. Thus, RHA is a multivalent extract with insecticide and antioxidant properties at low and safe concentrations. However, others studies on its indirect toxic effects are ongoing to ensure the complete safety of RHA.

Pre-meal active video game playing increased subjective appetite but not food intake in children and adolescents.

OBJECTIVE: Sedentary video game playing (VGP) and caloric preloads in the pre-meal environment have been shown to influence short-term food intake (FI) regulation in children. Other factors that may affect FI control include physical activity and/or heightened emotion. Therefore, we examined the effects of a glucose preload and 30 min of active VGP (aVGP) on subjective appetite, short-term FI, and subjective emotions in 9-14 year-old children. METHODS: On four test mornings approximately one-week apart, twenty-seven children (sex: 15M, 12F; age: 11.3 ± 0.3 years; BMI percentile: 55.3 ± 6.1%) consumed a standardized breakfast two hours prior to consuming 250 mL of either a 50 g glucose preload or Sucralose® control. Following the preload, participants participated in 30 min of quiet sitting or aVGP. Energy expenditure was measured during aVGP via indirect calorimetry. FI from an ad libitum pizza meal was measured after each test condition. Subjective appetite and emotions were measured at baseline (0 min), during treatment (15min), and immediately before the test meal (30 min). RESULTS: aVGP did not affect FI, but the glucose preload decreased FI compared with the sucralose control (∆ = 157 kcal, <0.001). Although not statistically significant (p=0.12), caloric compensation was lower following the glucose preload in the aVGP condition. Subjective appetite increased with time, and was higher in the sucralose control + aVGP condition (p=0.05). Change from baseline subjective emotion scores of anger and excitement were higher (p=0.03) and lower (p=0.02) after aVGP, respectively. CONCLUSIONS: Neither short-term FI nor net energy balance were affected by low-intensity aVGP (energy expenditure of 34 kcal). These findings suggest that a short bout of low-intensity aVGP does not alter energy balance during the study measurement period, and may not support achieving or maintaining healthy weights in children. However, future longitudinal studies are needed to confirm such advice.

Mesenchymal stem cells from sternum: the type of heart disease, ischemic or valvular, does not influence the cell culture establishment and growth kinetics.

BACKGROUND: In an attempt to increase the therapeutic potential for myocardial regeneration, there is a quest for new cell sources and types for cell therapy protocols. The pathophysiology of heart diseases may affect cellular characteristics and therapeutic results. METHODS: To study the proliferative and differentiation potential of mesenchymal stem cells (MSC), isolated from bone marrow (BM) of sternum, we made a comparative analysis between samples of patients with ischemic (IHD) or non-ischemic valvular (VHD) heart diseases. We included patients with IHD (n = 42) or VHD (n = 20), with average age of 60 years and no differences in cardiovascular risk factors. BM samples were collected (16.4 ± 6 mL) and submitted to centrifugation with Ficoll-Paque, yielding 4.5 ± 1.5 × 107 cells/mL. RESULTS: Morphology, immunophenotype and differentiation ability had proven that the cultivated sternal BM cells had MSC features. The colony forming unit-fibroblast (CFU-F) frequency was similar between groups (p = 0.510), but VHD samples showed positive correlation to plated cells vs. CFU-F number (r = 0.499, p = 0.049). The MSC culture was established in 29% of collected samples, achieved passage 9, without significant difference in expansion kinetics between groups (p > 0.05). Dyslipidemia and the use of statins was associated with culture establishment for IHD patients (p = 0.049 and p = 0.006, respectively). CONCLUSIONS: Together, these results show that the sternum bone can be used as a source for MSC isolation, and that ischemic or valvular diseases do not influence the cellular yield, culture establishment or in vitro growth kinetics.

Clinical performance of topical sodium fluoride when supplementing carbamide peroxide at-home bleaching gel.

This clinical study evaluated the use of 0.11% topical sodium fluoride (SF) desensitizing agent to treat tooth sensitivity during a nightguard tooth whitening procedure. Thirty-two subjects bleached their teeth with 10% carbamide peroxide (CP) gel using an at-home bleaching technique with custom trays. During bleaching treatment, subjects were divided into 2 groups (n = 16). The subjects in Group 1 received a topical gel containing 0.11% SF; the subjects in Group 2 received a placebo gel (PG). Each subject was instructed to place the gel in his/her bleaching tray for 30 min every day following bleaching treatment. Results showed the use of SF did not affect the whitening efficacy of the 10% CP gel. Subjects who received the PG had significantly higher tooth sensitivity when compared with subjects who received SF (P < 0.00). The use of daily 0.11% SF after 10% CP bleaching gel reduced tooth sensitivity during the bleaching treatment.

Bond strength of adhesive systems with different solvents to dry and wet dentin.

AIM: This study evaluates bond strength between dentin and composite using adhesives with different solvents to dry and wet dentin. MATERIALS AND METHODS: Ninety bovine incisors were used; the vestibular surfaces were worn by the exposure of an area with a diameter of 4 mm of dentin. The specimens were divided into 6 groups, according to the type of adhesive used and hydratation stals: Group SB-wet: Single Bond 2 in wet dentin, Group SBdry: Single Bond 2 in dry dentin, Group SL-wet: Solobond M in wet dentin, Group SL-dry: Solobond M in dentin dry. Group XPwet: XP Bond in wet dentin, Group XP-dry: XP Bond in dentin dry. They were cut to obtain specimens in the shape of stick with 1 × 1 mm and subjected to microtensile test in universal testing machine with a cross speed of 1mm/min. The data were analyzed with ANOVA and Tukey's tests (5%). RESULTS: ANOVA showed significant differences for surface treatment and interaction, but no difference was found for adhesive factor. The Tukey's test showed that the samples with wet dentin shown higher values of bond strength. CONCLUSION: The adhesive did not influence in the bond strength. The groups with wet dentin showed higher values of bond strength than groups with dry dentin.

Influence of CVD diamond tips and Er:YAG laser irradiation on bonding of different adhesive systems to dentin.

AIM: The aim of this study was to compare the microtensile bond strength of three adhesive systems, using different methods of dentin preparation. MATERIALS AND METHODS: A hundred and eight bovine teeth were used. The dentin from buccal face was exposed and prepared with three different methods, divided in 3 groups: Group 1 (DT)- diamond tip on a high-speed handpiece; Group 2 (CVD)-CVD tip on a ultrasonic handpiece; Group 3 (LA)-Er: YAG laser. The teeth were divided into 3 subgroups, according adhesive systems used: Subgroup 1-Adper Single Bond Plus/3M ESPE (SB) total-etch adhesive; Subgroup 2-Adper Scotchbond SE/3M ESPE (AS) selfetching adhesive; Subgroup 3-Clearfil SE Bond/Kuraray (CS) selfetching adhesive. Blocks of composite (Filtek Z250-3M ESPE) 4 mm high were built up and specimens were stored in deionized water for 24 hours at 37°C. Serial mesiodistal and buccolingual cuts were made and stick-like specimens were obtained, with transversal section of 1.0 mm(2). The samples were submitted to microtensile test at 1 mm/min and load of 10 kg in a universal testing machine. Data (MPa) were subjected to ANOVA and Tukey's tests (p < 0.05). RESULTS AND CONCLUSION: Surface treatment with Diamond or CVD tips associated with Clearfil SE Bond adhesive produced significantly lower bond strength values compared to other groups. Surface treatment with Er: YAG laser associated with Single Bond Plus or Clearfil SE Bond adhesives and surface treatment with CVD tip associated with Adper Scotchbond SE adhesive produced significantly lower bond strength values compared to surface treatment with diamond or CVD tips associated with Single Bond Plus or Adper Scotchbond SE adhesives. CLINICAL SIGNIFICANCE: Interactions between laser and the CVD tip technologies and the different adhesive systems can produce a satisfactory bonding strength result, so that these associations may be beneficial and enhance the clinical outcomes.

Bioinformatics for analysis of poxvirus genomes.

In recent years, there have been numerous unprecedented technological advances in the field of molecular biology; these include DNA sequencing, mass spectrometry of proteins, and microarray analysis of mRNA transcripts. Perhaps, however, it is the area of genomics, which has now generated the complete genome sequences of more than 100 poxviruses, that has had the greatest impact on the average virology researcher because the DNA sequence data is in constant use in many different ways by almost all molecular virologists. As this data resource grows, so does the importance of the availability of databases and software tools to enable the bench virologist to work with and make use of this (valuable/expensive) DNA sequence information. Thus, providing researchers with intuitive software to first select and reformat genomics data from large databases, second, to compare/analyze genomics data, and third, to view and interpret large and complex sets of results has become pivotal in enabling progress to be made in modern virology. This chapter is directed at the bench virologist and describes the software required for a number of common bioinformatics techniques that are useful for comparing and analyzing poxvirus genomes. In a number of examples, we also highlight the Viral Orthologous Clusters database system and integrated tools that we developed for the management and analysis of complete viral genomes.

Absence or weak correlation between chewing side preference and lateralities in primary, mixed and permanent dentition.

OBJECTIVES: To determine whether chewing side preference (CSP) is correlated to lateralities (handedness, footedness, eyedness and earedness) in primary, mixed and permanent dentitions. DESIGN: Three-hundred subjects were divided into 3 groups: Group 1--100 children 3-5 years old, primary dentition; Group 2--100 children 6-12 years old, mixed dentition; Group 3--100 subjects 18-47 years old, permanent dentition. CSP was determined using a method developed by Mc Donnell et al. Subjects were given a piece of gum and the position of the chewing gum was recorded 7 times as right or left. Subjects were classified as 'observed preferred chewing side' (OPCS) when they performed 5/7, 6/7 or 7/7 strokes on the same side. OPCS corresponded to the CSP. Laterality tests were performed for handedness, footedness, eyedness and earedness tasks. The Chi-square (χ(2)) and phi correlation (r) tests were used to investigate significant correlations between CSP and sidedness. RESULTS: There was a significant correlation between chewing and earedness (p=0.00), although there was weak positive correlation (r=0.30) for primary dentition. There were significant correlations between chewing and handedness (p=0.02; r=0.25) and chewing and footedness (p=0.02; r=0.26), however, there were weak positive correlations for mixed dentition; there were significant correlations between chewing and handedness (p=0.02; r=0.26); chewing and footedness (p=0.00; r=0.33) and chewing and earedness (p=0.01; r=0.29); however, there were weak positive correlations for permanent dentition. CONCLUSION: It may be concluded that CSP can be significantly correlated with: earedness for primary dentition; handedness and footedness for mixed dentition; handedness, footedness and earedness for permanent dentition, but these are weak positive relationships. Future work on larger samples of left- and right-sided individuals is required to validate the findings.

Evaluation of the cohesive strength between resin composite and light-curing characterizing materials.

PURPOSE: To evaluate the cohesive strength between composite and different light-curing characterizing materials (LCCM), which were prepared using the intrinsic technique. MATERIALS AND METHODS: One hundred composite specimens were made by using a prefabricated Teflon device, and a layer of LCCM was applied at the interface. The specimens were divided into 5 groups (n = 20): group 1 (control), no LCCM was used; group 2: application of White Kolor Plus Pigment (Kerr) LCCM; group 3: White Tetric Color Pigment (Ivoclar/Vivadent) LCCM; group 4: Brown Kolor Plus Pigment (Kerr) LCCM; group 5: Black Tetric Color Pigment (Ivoclar/Vivadent) LCCM. All materials were used according to the manufacturers' instructions. Specimens were submitted to a tensile test in a universal testing machine (EMIC DL-200MF) to evaluate the cohesive strength at the composite interface. Data were subjected to one-way ANOVA and Tukey's test (α = 5%). RESULTS: ANOVA showed a p-value = 0.0001, indicating that there were significant differences among the groups. The mean values in MPa (± standard deviation) obtained for the groups were: G1: 28.5 (± 2.74)a; G2: 23.5 (± 2.47)b; G3: 20.3 (± 2.49)b; G4: 10.5 (± 2.40)c; G5: 9.66 (± 3.06)c. The groups with the same letters presented no significant differences. The control group presented statistically significantly higher cohesive strengths when compared to the other groups. The groups in which Brown Kolor Plus Pigment and Black Tetric Color Pigment LCCM were used showed significantly lower cohesive strengths when compared to the groups in which White Kolor Plus Pigment and White Tetric Color Pigment LCMM were used. CONCLUSION: The use of LCCM produced with the intrinsic technique reduced the cohesive strength of composite.

Genomic analysis of highly virulent Georgia 2007/1 isolate of African swine fever virus.

African swine fever is widespread in Africa but has occasionally been introduced into other continents. In June 2007, African swine fever was isolated in the Caucasus Region of the Republic of Georgia and subsequently in neighboring countries (Armenia, Azerbaijan, and 9 states of the Russian Federation). Previous data for sequencing of 3 genes indicated that the Georgia 2007/1 isolate is closely related to isolates of genotype II, which has been identified in Mozambique, Madagascar, and Zambia. We report the complete genomic coding sequence of the Georgia 2007/1 isolate and comparison with other isolates. A genome sequence of 189,344 bp encoding 166 open reading frames (ORFs) was obtained. Phylogeny based on concatenated sequences of 125 conserved ORFs showed that this isolate clustered most closely with the Mkuzi 1979 isolate. Some ORFs clustered differently, suggesting that recombination may have occurred. Results provide a baseline for monitoring genomic changes in this virus.

Microshear bond strength of self-etching bonding systems to ultrasound diamond bur-prepared dentin.

PURPOSE: The aim of this study was to evaluate the influence of dentin abrasion on the microshear bond strength of two self-etching adhesive systems, using either an ultrasound diamond bur or a high-speed diamond bur. MATERIALS AND METHODS: Twenty noncarious human third molars were sectioned mesiodistally into halves. The enamel was ground to expose a flat dentin surface on both sections. The dentinal surfaces were randomly assigned to two groups, depending on the method of smear layer preparation: ultrasound diamond bur (UB) or conventional diamond bur (CB). The prepared dentin surfaces received one of two self-etching systems: Clearfil SE Bond (CF) and One-Up Bond F (OB). A composite cylinder with a 0.95-mm diameter was bonded to each specimen and the microshear bond test was performed. The results were expressed in MPa and were subjected to two-way analysis of variance (ANOVA) and Tukey's test (α = 0.05). RESULTS: There was no significant difference in dentin bond strength when comparing the conventional and ultrasonic abrasion methods. When the adhesive systems were compared, Clearfil SE Bond achieved higher bond strength means than did One-Up Bond F. CONCLUSION: The dentin surface preparation method did not influence the microshear bond strength and the Clearfil SE Bond adhesive system, independent of bur type used, Clearfil SE Bond showed higher bond strengths than did the One-Up Bond F adhesive system.

Prevalence of chewing side preference in the deciduous, mixed and permanent dentitions.

AIM: This study evaluated the prevalence of the chewing side preference (CSP) in the deciduous, mixed and permanent dentitions. MATERIALS AND METHODS: Three-hundred subjects were divided in three groups (n = 100): Group 1 - children with deciduous dentition; group 2-children with mixed dentition; group 3 - subjects with permanent dentition. The CSP was determined using a direct method (visual observation) developed by McDonnell et al. (2004). Descriptive statistic was used to observe the prevalence of CSP. The Pearson's Chi-square was used to investigate signicant associations between gender, presence of CSP and preferred side (right/left). RESULTS AND CONCLUSION: Eighty-seven percent of group 1 had a CSP. Eighty-two percent of group 2 had a CSP. Seventy-six percent of group 3 had a CSP. There was no statistically significant association between presence of CSP and gender in all groups. There was no statistically significant association between preferred side (right/left) and gender in all groups. CLINICAL SIGNIFICANCE: There is a higher prevalence of subjects in deciduous, mixed and permanent dentition that presented chewing side preference. The early diagnosis of the presence of chewing side preference can prevent the unilateral chewing pattern with prophylactic therapy applied to the first teeth (deciduous).

Phylogenomic analysis of 11 complete African swine fever virus genome sequences.

Viral molecular epidemiology has traditionally analyzed variation in single genes. Whole genome phylogenetic analysis of 123 concatenated genes from 11 ASFV genomes, including E75, a newly sequenced virulent isolate from Spain, identified two clusters. One contained South African isolates from ticks and warthog, suggesting derivation from a sylvatic transmission cycle. The second contained isolates from West Africa and the Iberian Peninsula. Two isolates, from Kenya and Malawi, were outliers. Of the nine genomes within the clusters, seven were within p72 genotype 1. The 11 genomes sequenced comprised only 5 of the 22 p72 genotypes. Comparison of synonymous and non-synonymous mutations at the genome level identified 20 genes subject to selection pressure for diversification. A novel gene of the E75 virus evolved by the fusion of two genes within the 360 multicopy family. Comparative genomics reveals high diversity within a limited sample of the ASFV viral gene pool.

Vaccinia virus G8R protein: a structural ortholog of proliferating cell nuclear antigen (PCNA).

BACKGROUND: Eukaryotic DNA replication involves the synthesis of both a DNA leading and lagging strand, the latter requiring several additional proteins including flap endonuclease (FEN-1) and proliferating cell nuclear antigen (PCNA) in order to remove RNA primers used in the synthesis of Okazaki fragments. Poxviruses are complex viruses (dsDNA genomes) that infect eukaryotes, but surprisingly little is known about the process of DNA replication. Given our previous results that the vaccinia virus (VACV) G5R protein may be structurally similar to a FEN-1-like protein and a recent finding that poxviruses encode a primase function, we undertook a series of in silico analyses to identify whether VACV also encodes a PCNA-like protein. RESULTS: An InterProScan of all VACV proteins using the JIPS software package was used to identify any PCNA-like proteins. The VACV G8R protein was identified as the only vaccinia protein that contained a PCNA-like sliding clamp motif. The VACV G8R protein plays a role in poxvirus late transcription and is known to interact with several other poxvirus proteins including itself. The secondary and tertiary structure of the VACV G8R protein was predicted and compared to the secondary and tertiary structure of both human and yeast PCNA proteins, and a high degree of similarity between all three proteins was noted. CONCLUSIONS: The structure of the VACV G8R protein is predicted to closely resemble the eukaryotic PCNA protein; it possesses several other features including a conserved ubiquitylation and SUMOylation site that suggest that, like its counterpart in T4 bacteriophage (gp45), it may function as a sliding clamp ushering transcription factors to RNA polymerase during late transcription.

Genomic differences of Vaccinia virus clones from Dryvax smallpox vaccine: the Dryvax-like ACAM2000 and the mouse neurovirulent Clone-3.

Conventional vaccines used for smallpox eradication were often denoted one or another strain of Vaccinia virus (VACV), even though seed virus was sub-cultured multifariously, which rendered the virion population genetically heterogeneous. ACAM2000 cell culture vaccine, recently licensed in the U.S., consists of a biologically vaccine-like VACV homogeneous-sequence clone from the conventional smallpox vaccine Dryvax, which we verified from Dryvax sequence chromatograms is genetically heterogeneous. ACAM2000 VACV and CL3, a mouse-neurovirulent clone from Dryvax, differ by 572 single nucleotide polymorphisms and 53 insertions-deletions of varied size, including a 4.5-kbp deletion in ACAM2000 and a 6.2-kbp deletion in CL3. The sequence diversity between the two clones precludes precisely defining why CL3 is more pathogenic; however, four genes appear significantly dissimilar to account for virulence differences. CL3 encodes intact immunomodulators interferon-alpha/beta and tumor necrosis factor receptors, which are truncated in ACAM2000. CL3 specifies a Cowpox and Variola virus-like ankyrin-repeat protein that might be associated with proteolysis via ubiquitination. And, CL3 shows an elongated thymidylate kinase, similar to the enzyme of the mouse-neurovirulent VACV-WR, a derivative of the New York City Board of Health vaccine, the origin vaccine of Dryvax. Although ACAM2000 encodes most proteins associated with immunization protection, the cloning probably delimited the variant epitopes and other motifs produced by Dryvax due to its VACV genetic heterogeneity. The sequence information for ACAM2000 and CL3 could be significant for resolving the dynamics of their different proteomes and thereby aid development of safer, more effective vaccines.

An ectromelia virus profilin homolog interacts with cellular tropomyosin and viral A-type inclusion protein.

BACKGROUND: Profilins are critical to cytoskeletal dynamics in eukaryotes; however, little is known about their viral counterparts. In this study, a poxviral profilin homolog, ectromelia virus strain Moscow gene 141 (ECTV-PH), was investigated by a variety of experimental and bioinformatics techniques to characterize its interactions with cellular and viral proteins. RESULTS: Profilin-like proteins are encoded by all orthopoxviruses sequenced to date, and share over 90% amino acid (aa) identity. Sequence comparisons show highest similarity to mammalian type 1 profilins; however, a conserved 3 aa deletion in mammalian type 3 and poxviral profilins suggests that these homologs may be more closely related. Structural analysis shows that ECTV-PH can be successfully modelled onto both the profilin 1 crystal structure and profilin 3 homology model, though few of the surface residues thought to be required for binding actin, poly(L-proline), and PIP2 are conserved. Immunoprecipitation and mass spectrometry identified two proteins that interact with ECTV-PH within infected cells: alpha-tropomyosin, a 38 kDa cellular actin-binding protein, and the 84 kDa product of vaccinia virus strain Western Reserve (VACV-WR) 148, which is the truncated VACV counterpart of the orthopoxvirus A-type inclusion (ATI) protein. Western and far-western blots demonstrated that the interaction with alpha-tropomyosin is direct, and immunofluorescence experiments suggest that ECTV-PH and alpha-tropomyosin may colocalize to structures that resemble actin tails and cellular protrusions. Sequence comparisons of the poxviral ATI proteins show that although full-length orthologs are only present in cowpox and ectromelia viruses, an ~ 700 aa truncated ATI protein is conserved in over 90% of sequenced orthopoxviruses. Immunofluorescence studies indicate that ECTV-PH localizes to cytoplasmic inclusion bodies formed by both truncated and full-length versions of the viral ATI protein. Furthermore, colocalization of ECTV-PH and truncated ATI protein to protrusions from the cell surface was observed. CONCLUSION: These results suggest a role for ECTV-PH in intracellular transport of viral proteins or intercellular spread of the virus. Broader implications include better understanding of the virus-host relationship and mechanisms by which cells organize and control the actin cytoskeleton.

Predicted function of the vaccinia virus G5R protein.

MOTIVATION: Of the approximately 200 proteins that have been identified for the vaccinia virus (VACV) genome, many are currently listed as having an unknown function, and seven of these are also found in all other poxvirus genomes that have been sequenced. The G5R protein of VACV is included in this list, and to date, very little is known about this essential and highly conserved protein. Conventional similarity searches of protein databases do not identify significantly similar proteins, and experimental approaches have been unsuccessful at determining protein function. RESULTS: Using HHsearch, a hidden Markov model (HMM) comparison search tool, the G5R protein was found to be similar to both human and archaeal flap endonucleases (FEN-1) with 96% probability. The G5R protein structure was subsequently successfully modeled using the Robetta protein structure prediction server with an archaeal FEN-1 as the template. The G5R model was then compared to the human FEN-1 crystal structure and was found to be structurally similar to human FEN-1 in both active site residues and DNA substrate binding regions.

New bioinformatics tools for viral genome analyses at Viral Bioinformatics--Canada.

Viruses are much smaller than prokaryotes and eukaryotes, and it is now practical to sequence closely related members of virus families, strains, or even different isolates recovered during the course of an outbreak. However, comparative analysis of viral genomes requires the development of novel bioinformatics tools that allow us to align, edit, compare and interact with these genomes at all levels, from whole genome, to gene family, to single nucleotide polymorphisms. Comparative viral genomics can lead to the identification of the core characteristics that define a virus family, as well as the unique properties of viral species or isolates that contribute to variations in pathogenesis. This paper describes a number of tools, mainly developed for Viral Bioinformatics--Canada, that can be used for annotation and comparative genomic analysis of poxviruses. Nonetheless, these tools are also broadly applicable to other virus families.

Host-derived pathogenicity islands in poxviruses.

BACKGROUND: Poxviruses are important both as pathogens and as vaccine vectors. Poxvirus genomes (150-350 kb) consist of a single linear dsDNA molecule; the two polynucleotide strands are joined by short hairpin loops. The genomes encode highly conserved proteins required for DNA replication and mRNA transcription as well as a variable set of virulence factors; transcription takes place within the cytoplasm of the host cell. We are interested in evolution of poxvirus genomes and especially how these viruses acquire host-derived genes that are believed to function as virulence factors. RESULTS: Using a variety of bioinformatics tools, we have identified regions in poxvirus genomes that have unusual nucleotide composition (higher or lower than average A+T content) compared to the genome as a whole; such regions may be several kilobases in length and contain a number of genes. Regions with unusual nucleotide composition may represent genes that have been recently acquired from the host genome. The study of these genomic regions with unusual nucleotide content will help elucidate evolutionary processes in poxviruses. CONCLUSION: We have found that dotplots of complete poxvirus genomes can be used to locate regions on the genome that differ significantly in A+T content to the genome as a whole. The genes in these regions may have been acquired relatively recently from the host genome or from another AT-rich poxvirus.