Shigella flexneri is the major Shigella species that causes diarrheal disease in developing countries. It is further subdivided into 15 serotypes based on O-antigen structure. Serotyping of S. flexneri is important for epidemiological purposes. In this study, we developed a multiplex PCR assay targeting the O-antigen synthesis gene wzx and the O-antigen modification genes gtrI, gtrIC, gtrII, oac, gtrIV, gtrV, and gtrX for molecular serotyping of S. flexneri. The multiplex PCR assay contained eight sets of specific PCRs in a single tube and can identify 14 of the 15 serotypes (the exception being serotype Xv) of S. flexneri recognized thus far. A nearly perfect concordance (97.8%) between multiplex PCR assay and slide agglutination was observed when 358 S. flexneri strains of various serotypes were analyzed, except that 8 strains were carrying additional cryptic and/or defective serotype-specific genes. The multiplex PCR assay provides a rapid and specific method for the serotype identification of S. flexneri.
The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.
Shigella spp. are the causative agent of shigellosis with Shigella flexneri serotype 2a being the most prevalent in developing countries. Epidemiological surveillance in China found that a new serotype of S. flexneri appeared in 2001 and replaced serotype 2a in 2003 as the most prevalent serotype in Henan Province. The new serotype also became the dominant serotype in 7 of the 10 other provinces under surveillance in China by 2007. The serotype was identified as a variant of serotype X. It differs from serotype X by agglutination to the monovalent anti-IV type antiserum and the group antigen-specific monoclonal antibody MASF IV-I. Genome sequencing of a serotype X variant isolate, 2002017, showed that it acquired a Shigella serotype conversion island, also as an SfX bacteriophage, containing gtr genes for type X-specific glucosylation. Multilocus sequence typing of 15 genes from 37 serotype X variant isolates and 69 isolates of eight other serotypes, 1a, 2a, 2b, 3a, 4a, 5b, X, and Y, found that all belong to a new sequence type (ST), ST91. Pulsed-field gel electrophoresis revealed 154 pulse types with 655 S. flexneri isolates analyzed and identified 57 serotype switching events. The data suggest that S. flexneri epidemics in China have been caused by a single epidemic clone, ST91, with frequent serotype switching to evade infection-induced immunity to serotypes to which the population was exposed previously. The clone has also acquired resistance to multiple antibiotics. These findings underscore the challenges to the current vaccine development and control strategies for shigellosis.
The sequence of the nonredundant region of the Salmonella enterica serovar Typhimurium temperate, serotype-converting bacteriophage P22 has been completed. The genome is 41,724 bp with an overall moles percent GC content of 47.1%. Numerous examples of potential integration host factor and C1-binding sites were identified in the sequence. In addition, five potential rho-independent terminators were discovered. Sixty-five genes were identified and annotated. While many of these had been described previously, we have added several new ones, including the genes involved in serotype conversion and late control. Two of the serotype conversion gene products show considerable sequence relatedness to GtrA and -B from Shigella phages SfII, SfV, and SfX. We have cloned the serotype-converting cassette (gtrABC) and demonstrated that it results in Salmonella serovar Typhimurium LT2 cells which express antigen O1. Many of the putative proteins show sequence relatedness to proteins from a great variety of other phages, supporting the hypothesis that this phage has evolved through the recombinational exchange of genetic information with other viruses.
The O-antigen of Salmonella lipopolysaccharide is a major antigenic determinant and its chemical composition forms the basis for Salmonella serotyping. Modifications of the O-antigen that can affect the serotype include those carried out by the products of glycosyltransferase operons (gtr), which are present on specific Salmonella and phage genomes. Here we show that expression of the gtr genes encoded by phage P22 that confers the O1 serotype is under the control of phase variation. This phase variation occurs by a novel epigenetic mechanism requiring OxyR in conjunction with the DNA methyltransferase Dam. OxyR is an activator or a repressor of the system depending on which of its two binding sites in the gtr regulatory region is occupied. Binding is decreased by methylation at Dam target sequences in either site, and this confers heritability of the expression state to the system. Most Salmonella gtr operons share the key regulatory elements that are identified here as essential for this epigenetic phase variation.
The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions.
Bacterial pathogens frequently evolve mechanisms to vary the composition of their surface structures. The consequence is enhanced long-term survival by facilitating persistence and evasion of the host immune system. Salmonella sp., cause severe infections in a range of mammalian hosts and guard themselves with a protective coat, termed the O-antigen. Through genome sequence analyses we found that Salmonella have acquired an unprecedented repertoire of genetic sequences for modifying their O-antigen coat. There is strong evidence that these genetic factors have a dynamic evolutionary history and are spread through the bacterial population by bacteriophage. In addition to this genetic repertoire, we determined that Salmonella can and often do employ stochastic mechanisms for expression of these genetic factors. This means that O-antigen coat diversity can be generated within a Salmonella population that otherwise has a common genome. Our data significantly enhance our appreciation of the genetic and regulatory characteristics underpinning Salmonella O-antigen diversity. The role attributed to bacteriophage in generating this diversity highlights that Salmonella are acquiring an extensive repertoire of O-antigen modifying traits that may enhance the pathogen's ability to persist and cause disease in mammalian hosts. Such genetic traits may make useful markers for defining new epidemiological and diagnostic tools.
All Shigella flexneri serotypes except serotype 6 share a common O-antigen tetrasaccharide backbone and nearly all variations between serotypes are due to glucosyl and/or O-acetyl modifications of the common O unit mediated by glycosyltransferases encoded by serotype-converting bacteriophages. Several S. flexneri serotype-converting phages including SfV, SfX, Sf6 and SfII have been isolated and characterized. However, S. flexneri serotype-converting phage SfI which encodes a type I modification of serotype 1 (1a, 1b, 1c and 1d) had not yet been characterized.
The SfI phage was induced and purified from a S. flexneri serotype 1a clinical strain 019. Electron microscopy showed that the SfI phage has a hexagonal head and a long contractile tail, characteristic of the members of Myoviridae family. SfI can convert serotype Y to serotype 1a and serotype X to serotype 1d, but cannot convert 10 other S. flexneri serotypes (1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, Xv) tested, suggesting that SfI has a narrow host range. Similar to other S. flexneri serotype-converting phages, SfI integrates into the tRNA-thrW gene adjacent to proA of the host chromosome when lysogenized. The complete sequence of the SfI genome was 38,389 bp, encoding 66 open reading frames and two tRNA genes. Phage SfI shares significant homology with S. flexneri phage SfV, Escherichia coli prophage e14 and lambda, and is classified into the lambdoid phage family. SfI was found to use a cos mechanism for DNA packaging similar to that of phage SfV.
SfI contains features of lambdoid phages and is closely related to S. flexneri phage SfV, E. coli prophage e14 and lambda. The characterization of SfI enhances our understanding of serotype conversion of S. flexneri.
We have found an open reading frame which is 1.1 kb upstream of PHO84 (which encodes a Pi transporter) and is transcribed from the opposite strand. In Saccharomyces cerevisiae, this gene is distal to the TUB3 locus on the left arm of chromosome XIII and is named GTR1. GTR1 encodes a protein consisting of 310 amino acid residues containing, in its N-terminal region, the characteristic tripartite consensus elements for binding GTP conserved in GTP-binding proteins, except for histidine in place of a widely conserved aspargine residue in element III. Disruption of the GTR1 gene resulted in slow growth at 30 degrees C and no growth at 15 degrees C; other phenotypes resembled those of pho84 mutants and included constitutive synthesis of repressible acid phosphatase, reduced Pi transport activity, and resistance to arsenate. The latter phenotypes were shown to be due to a defect in Pi uptake, and the Gtr1 protein was found to be functionally associated with the Pho84 Pi transporter. Recombination between chromosome V (at the URA3 locus) and chromosome XIII (in the GTR1-PHO84-TUB3 region) by using a plasmid-encoded site-specific recombination system indicated that the order of these genes was telomere-TUB3-PHO84-GTR1-CENXIII.
Background and Objectives:
The primary goal of periodontal therapy is to restore the tooth supporting tissues lost due to periodontal disease. The aim of the present study was to compare the efficacy of combination of type I collagen (GTR membrane) and xenogenic bone graft with open flap debridement (OFD) in treatment of periodontal intrabony defects.
Materials and Methods:
Twenty paired intrabony defects were surgically treated using split mouth design. The defects were randomly assigned to treatment with OFD + collagen membrane + bone graft (Test) or OFD alone (Control). The clinical efficacy of two treatment modalities was evaluated at 9 month postoperatively by clinical, radiographical, and intrasurgical (re-entry) parameters. The measurements included probing pocket depth (PD), clinical attachment level (CAL), gingival recession (GR), bone fill (BF), bone density (BD) and intra bony component (INTRA).
The mean reduction in PD at 0–9 month was 3.3±0.82 mm and CAL gain of 3.40±1.51 mm occurred in the collagen membrane + bone graft (Test) group; corresponding values for OFD (Control) were 2.20±0.63 mm and 1.90±0.57 mm. Similar pattern of improvement was observed when radiographical and intra-surgical (re-entry) post operative evaluation was made. All improvement in different parameters was statistically significant (P< 0.01).
Interpretation and Conclusion:
Treatment with a combination of collagen membrane and bone graft led to a significantly more favorable clinical outcome in intrabony defects as compared to OFD alone.
Bone grafts; collagen membrane; guided tissue regeneration; periodontal regeneration
The aim of this clinical trial was to evaluate the additional benefit of using guided tissue regeneration (GTR) with autogenous cortical bone (ACB) grafting versus ACB grafting alone for the regenerative treatment of intraosseous periodontal defects.
Via a split-mouth design, 12 patients with chronic periodontitis (five men, seven women; mean age, 45.3±4.6 years) who had probing pocket depths (PPDs) of ≥6 mm following initial periodontal therapy were randomly assigned to two treatments in contralateral areas of the dentition: a combination of ACB grafting and GTR (with a absorbable membrane of polylactic acid) or ACB grafting alone. The compared parameters were preoperative and 6-month postoperative PPDs, clinical attachment levels (CALs), and radiographic alveolar bone heights.
Both treatment modalities resulted in significant changes in the postoperative measurements from the preoperative values (P<.01). The reduction in the PPDs, gain in the CALs, and gain in the radiographic alveolar bone heights were 4.58±1.08, 4.25±1.06, and 5.50±2.24 mm in the patients treated with ACB grafting and GTR and 4.92±1.00, 4.50±0.80, and 5.92±1.83 mm in those treated with ACB grafting alone, respectively. The differences between the treatments were not statistically significant (P>.05).
Within the study limitations, both ACB grafting with GTR and ACB grafting alone lead to significant improvements in clinical and radiographic parameters at 6 months postoperatively. The combined approach does not provide any additional benefit for treating intraosseous periodontal defects.
Autogenous bone graft; Guided tissue regeneration; Intraosseous defects; Periodontal regeneration
Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium commonly used as a model organism for studying cyanobacterial cell differentiation and nitrogen fixation. For many decades, this cyanobacterium was considered an obligate photo-lithoautotroph. We now discovered that this strain is also capable of mixotrophic, photo-organoheterotrophic, and chemo-organoheterotrophic growth if high concentrations of fructose (at least 50 mM and up to 200 mM) are supplied. Glucose, a substrate used by some facultatively organoheterotrophic cyanobacteria, is not effective in Anabaena sp. PCC 7120. The gtr gene from Synechocystis sp. PCC 6803 encoding a glucose carrier was introduced into Anabaena sp. PCC 7120. Surprisingly, the new strain containing the gtr gene did not grow on glucose but was very sensitive to glucose, with a 5 mM concentration being lethal, whereas the wild-type strain tolerated 200 mM glucose. The Anabaena sp. PCC 7120 strain containing gtr can grow mixotrophically and photo-organoheterotrophically, but not chemo-organoheterotrophically with fructose. Anabaena sp. PCC 7120 contains five respiratory chains ending in five different respiratory terminal oxidases. One of these enzymes is a mitochondrial-type cytochrome c oxidase. As in almost all cyanobacteria, this enzyme is encoded by three adjacent genes called coxBAC1. When this locus was disrupted, the cells lost the capability for chemo-organoheterotrophic growth.
The prognosis for malignant gliomas remains dismal. We addressed the safety, feasibility and preliminary clinical activity of the vaccinations using autologous glioma cells and interleukin (IL)-4 gene transfected fibroblasts.
In University of Pittsburgh Cancer Institute (UPCI) protocol 95-033, adult participants with recurrent glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) received gross total resection (GTR) of the recurrent tumors, followed by two vaccinations with autologous fibroblasts retrovirally transfected with TFG-IL4-Neo-TK vector admixed with irradiated autologous glioma cells. In UPCI 99-111, adult participants with newly diagnosed GBM or AA, following GTR and radiation therapy, received two intradermal vaccinations with the TFG-IL4-Neo-TK-transfected fibroblasts admixed with type-1 dendritic cells (DC) loaded with autologous tumor lysate. The participants were evaluated for occurrence of adverse events, immune response, and clinical response by radiological imaging.
Results and Discussion
In UPCI 95-033, only 2 of 6 participants received the vaccinations. Four other participants were withdrawn from the trial because of tumor progression prior to production of the cellular vaccine. However, both participants who received two vaccinations demonstrated encouraging immunological and clinical responses. Biopsies from the local vaccine sites from one participant displayed IL-4 dose-dependent infiltration of CD4+ as well as CD8+ T cells. Interferon (IFN)-γ Enzyme-Linked Immuno-SPOT (ELISPOT) assay in another human leukocyte antigen (HLA)-A2+ participant demonstrated systemic T-cell responses against an HLA-A2-restricted glioma-associated antigen (GAA) epitope EphA2883–891. Moreover, both participants demonstrated clinical and radiological improvement with no evidence of allergic encephalitis, although both participants eventually succumbed with the tumor recurrence. In 99-111, 5 of 6 enrolled participants received scheduled vaccinations with no incidence of major adverse events. Monocyte-derived DCs produced high levels of IL-12 p70. Treatment was well tolerated; however, we were unable to observe detectable IFN-γ post-vaccine responses or prolonged progression-free survival in these participants.
Feasibility challenges inherent in the generation of a patient-specific gene transfection-based vaccine strongly suggests the need for more practical formulations that would allow for the timely administration of vaccines. Nevertheless, successful generation of type-1 DCs and preliminary safety in the current study provide a strong rationale for further efforts to develop novel glioma vaccines.
Most of the Shigella flexneri O-specific serotypes result from O-acetyl and/or glucosyl groups added to a common O-repeating unit of the lipopolysaccharide (LPS) molecule. The genes involved in acetylation and/or glucosylation of S. flexneri LPS are physically located on lysogenic bacteriophages, whereas the rfb cluster contains the biosynthesis genes for the common O-repeating unit (D.A.R. Simmons and E. Romanowska, J. Med. Microbiol. 23:289-302, 1987). Using a cosmid cloning strategy, we have cloned the rfb regions from S. flexneri 3a and 2a. Escherichia coli K-12 containing plasmids pYS1-5 (derived from S. flexneri 3a) and pEY5 (derived from S. flexneri 2a) expressed O-specific LPS which reacted immunologically with S. flexneri polyvalent O antiserum. However, O-specific LPS expressed in E. coli K-12 also reacted with group 6 antiserum, indicating the presence of O-acetyl groups attached to one of the rhamnose components of the O-repeating unit. This was confirmed by measuring the amounts of acetate released from purified LPS samples and also by the chemical removal of O-acetyl groups, which abolished group 6 reactivity. The O-acetylation phenotype was absent in an E. coli strain with an sbcB-his-rfb chromosomal deletion and could be restored upon conjugation of F' 129, which carries sequences corresponding to a portion of the deleted region. Our data demonstrate that E. coli K-12 strains possess a novel locus which directs the O acetylation of LPS and is located in the sbcB-rfb region of the chromosomal map.
Shigella flexneri is the major cause of shigellosis in the developing countries. The O-antigen component of the lipopolysaccharide is one of the key virulence determinants required for the pathogenesis of S. flexneri. The glucosyltransferase and/or acetyltransferase genes responsible for the modification of the O-antigen are encoded by temperate serotype converting bacteriophage present in the S. flexneri genome. Several serotype converting phages have previously been isolated and characterized, however, attempts to isolate a serotype converting phage which encodes the modification genes of serotypes 4a strain have not been successful.
In this study, a novel temperate serotype converting bacteriophage SfIV was isolated. Lysogenisation of phage SfIV converted serotype Y strain to serotype 4a. Electron microscopy indicated that SfIV belongs to Myoviridae family. The 39,758 bp genome of phage SfIV encompasses 54 open reading frames (orfs). Protein level comparison of SfIV with other serotype converting phages of S. flexneri revealed that SfIV is similar to phage SfII and SfV. The comparative analysis also revealed that SfIV phage contained five proteins which were not found in any other phages of S. flexneri. These proteins were: a tail fiber assembly protein, two hypothetical proteins with no clear function, and two other unknown proteins which were encoded by orfs present on a moron, that presumably got introduced in SfIV genome from another species via a transposon. These unique proteins of SfIV may play a role in the pathogenesis of the host.
This study reports the isolation and complete genome sequence analysis of bacteriophage SfIV. The SfIV phage has a host range significantly different from the other phages of Shigella. Comparative genome analysis identified several proteins unique to SfIV, which may potentially be involved in the survival and pathogenesis of its host. These findings will further our understanding on the evolution of these phages, and will also facilitate studies on development of new phage vectors and therapeutic agents to control infections caused by S. flexneri.
Shigella flexneri; Bacteriophage; O-antigen modification; Serotype conversion
Shigella flexneri is the major pathogen causing bacillary dysentery in developing countries. S. flexneri is divided into at least 16 serotypes based on the combination of antigenic determinants present in the O-antigen. All the serotypes (except for serotype 6) share a basic O-unit containing one N-acetyl-d-glucosamine and three l-rhamnose residues, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation. Serotype Xv is a newly emerged and the most prevalent serotype in China, which can agglutinate with both MASF IV-1 and 7,8 monoclonal antibodies. The factor responsible for the presence of MASF IV-1 (E1037) epitope has not yet been identified. In this study, we analyzed the LPS structure of serotype Xv strains and found that the MASF IV-1 positive phenotype depends on an O-antigen modification with a phosphoethanolamine (PEtN) group attached at position 3 of one of the rhamnose residues. A plasmid carried gene, lpt-O (LPS phosphoethanolamine transferase for O–antigen), mediates the addition of PEtN for serotype Xv and other MASF IV-1 positive strains. These findings reveal a novel serotype conversion mechanism in S. flexneri and show the necessity of further extension of the serotype classification scheme recognizing the MASF IV-1 positive strains as distinctive subtypes.
The Gtr1 protein of Saccharomyces cerevisiae is a member of the RagA subfamily of the Ras-like small GTPase superfamily. Gtr1 has been implicated in various cellular processes. Particularly, the Switch regions in the GTPase domain of Gtr1 are essential for TORC1 activation and amino acid signaling. Therefore, knowledge about the biochemical activity of Gtr1 is required to understand its mode of action and regulation.
By employing tryptophan fluorescence analysis and radioactive GTPase assays, we demonstrate that Gtr1 can adopt two distinct GDP- and GTP-bound conformations, and that it hydrolyses GTP much slower than Ras proteins. Using cysteine mutagenesis of Arginine-37 and Valine-67, residues at the Switch I and II regions, respectively, we show altered GTPase activity and associated conformational changes as compared to the wild type protein and the cysteine-less mutant.
The extremely low intrinsic GTPase activity of Gtr1 implies requirement for interaction with activating proteins to support its physiological function. These findings as well as the altered properties obtained by mutagenesis in the Switch regions provide insights into the function of Gtr1 and its homologues in yeast and mammals.
Gtr1; GTPase; Intrinsic tryptophan fluorescence; Rag GTPase; Cysteine mutagenesis; Switch region
Shigella flexneri is the major cause of bacterial shigellosis in developing countries. S. flexneri is divided into at least 19 serotypes, the majority of which are modifications of the same basic O-antigen by glucosylation and/or O-acetylation of its sugar residues by phage encoded serotype-converting genes. Recently, a plasmid encoded phosphoethanolamine (PEtN) modification of the O-antigen has been reported, which is responsible for the presence of the MASF IV-1 determinant and results in conversion of traditional serotypes X, 4a and Y to novel serotypes Xv, 4av and Yv, respectively. In this study, we characterized 19 serotype Yv strains isolated in China. A variant of the O-antigen phosphoethanolamine transferase gene opt (formerly called lpt-O) carried by a pSFxv_2-like plasmid was found in serotype Yv strains, which specifies the phosphorylation pattern on the O-antigen of this serotype. For the majority of the O-antigen units, the PEtN modification occurs on RhaIII, while for a minority, modifications occur on both RhaII and RhaIII. Serotype-specific gene detection and PFGE analysis suggested that these serotype Yv isolates were originated from serotypes Y, Xv and 2a by acquisition of an opt-carrying plasmid and/or inactivation of serotype-specific gene gtrII or gtrX. These data, combined with those of serotypes Xv and 4av reported earlier, demonstrate that the plasmid-encoded PEtN modification is an important serotype conversion mechanism in S. flexneri, in addition to glucosylation and O-acetylation.
Shigella flexneri causes diarrheal diseases especially in infants and children in developing countries. Modifications of lipopolysaccharide (LPS) molecule, like bacteriophage-mediated glucosylation and acetylation of the O-specific chain (O-SP), are important for the LPS antigenicity and consequently for the immunogenicity of the polysaccharide-based vaccines against shigellosis. Here we report the degree of O-acetylation and the localisation of O-acetyl groups and side-chain glucose substitution in the O-SP (scheme) in different preparations of S. flexneri type 2a LPS.
shigella; flexneri; LPS; O-acetylation; O-specific chain
Furcation invasions represent one of the most demanding therapeutic challenges in periodontics. This investigation assessed and compared the clinical efficacy of hydroxyapatite bone graft material when used alone and with collagen membrane in the treatment of grade II furcation defects.
Materials and Methods:
Ten patients with comparable bilateral furcation defects in relation to mandibular first molars were selected and treated in a split-mouth design. After the hygiene phase of therapy was completed, the groups were selected randomly either for treatment with hydroxyapatite bone graft (Periobone G) alone or with a combination of bone graft and guided tissue regeneration (GTR) membrane (Periocol). Clinical parameters like plaque index, gingival index, vertical probing depth, horizontal probing depth, clinical attachment level, position of marginal gingiva, and the amount of bone fill were used at baseline and at 3 and 6 months postoperatively.
At 6 months, both surgical procedures resulted in statistically significant reduction in vertical and horizontal probing depths and gain in the clinical attachment level.
The use of combination technique yielded superior results compared to sites treated with bone graft alone. However, the difference was not statistically significant.
Collagen; furcation defects; guided tissue regeneration; hydroxyapatite; surgical flaps
The present, randomized, controlled clinical and radiographic study was undertaken to compare the effectiveness of guided tissue regeneration (GTR) by using a collagen membrane barrier with or without decalcified freeze-dried bone allograft (DFDBA) in the treatment of periodontal infrabony defects characterized by unfavorable architecture.
Materials and Methods:
Sixteen systemically healthy patients with 20 periodontal infrabony defects were selected for the study. Each patient had at least ≥ 5 mm clinical probing pocket depth (PPD) at the selected site and depth of intrabony component ≥ 3 mm as assessed by clinical and radiographic measurements. Baseline measurements included plaque index, papillary bleeding index, PPD, gingival recession, clinical attachment level and radiographic defect depth (DD). At the time of surgery, the defects were randomly assigned to either the test group (collagen membrane plus DFDBA) or the control group (collagen membrane only).
At the 6-month examination, PPPD reduction was significantly greater in the GTR + DFDBA group (4.06 ± 0.38 mm) compared with the GTR group (3.2 ± 0.74 mm). The mean gains of clinical attachment were 3.54 ± 0.36 mm in the test group and 2.50 ± 0.74 mm in the control group. Radiographic DD reduction was similarly greater in the GTR + DFDBA group (2.40 ± 0.51 mm) compared with the GTR group (1.60 ± 0.51 mm).
The results of the present study indicate that the use of a GTR membrane with bone graft has significantly improved all clinical parameters tested as compared with the use of bioresorbable membrane alone in the treatment of infrabony defects characterized by unfavorable architecture.
Bone graft; guided tissue regeneration; infrabony defect
Three new species of Pythium, namely, P. oopapillum, P. emineosum and P. camurandrum are presented in this paper based on morphological descriptions and molecular phylogenetic characterisation. These new species were isolated from various ecological regions in Canada. They have unique morphological features in the genus Pythium, and form distinct clades in maximum parsimony analyses, which are also supported by maximum likelihood phylogeny using general time reversible model (GTR), and Bayesian inference (BI) phylogeny using Markov Chain Monte Carlo (MCMC) analysis methods. A comparative study of the new species with closely related taxa, their clade positions, and morphological features are described in this paper.
COI; ITS rDNA; LSU rDNA; Oomycete; Peronosporales; Pythium; taxonomy
Different types of bioabsorbable and nonresorbable membranes have been widely used for guided tissue regeneration (GTR) with its ultimate goal of regenerating lost periodontal structures. The purpose of the present study was to evaluate the biological effects of various bioabsorbable and nonresorbable membranes in cultures of primary human gingival fibroblasts (HGF), periodontal ligament fibroblasts (PDLF) and human osteoblast-like (HOB) cells in vitro.
Three commercially available collagen membranes [TutoDent® (TD), Resodont® (RD) and BioGide® (BG)] as well as three nonresorbable polytetrafluoroethylene (PTFE) membranes [ACE (AC), Cytoplast® (CT) and TefGen-FD® (TG)] were tested. Cells plated on culture dishes (CD) served as positive controls. The effect of the barrier membranes on HGF, PDLF as well as HOB cells was assessed by the Alamar Blue fluorometric proliferation assay after 1, 2.5, 4, 24 and 48 h time periods. The structural and morphological properties of the membranes were evaluated by scanning electron microscopy (SEM).
The results showed that of the six barriers tested, TD and RD demonstrated the highest rate of HGF proliferation at both earlier (1 h) and later (48 h) time periods (P < 0.001) compared to all other tested barriers and CD. Similarly, TD, RD and BG had significantly higher numbers of cells at all time periods when compared with the positive control in PDLF culture (P ≤ 0.001). In HOB cell culture, the highest rate of cell proliferation was also calculated for TD at all time periods (P < 0.001). SEM observations demonstrated a microporous structure of all collagen membranes, with a compact top surface and a porous bottom surface, whereas the nonresorbable PTFE membranes demonstrated a homogenous structure with a symmetric dense skin layer.
Results from the present study suggested that GTR membrane materials, per se, may influence cell proliferation in the process of periodontal tissue/bone regeneration. Among the six membranes examined, the bioabsorbable membranes demonstrated to be more suitable to stimulate cellular proliferation compared to nonresorbable PTFE membranes.
In periodontology, Guided Tissue Regeneration (GTR) is based on the concept of providing a space for entry of cells with regenerative potential into the wound environment to initiate the regeneration of structures lost due to periodontal disease. First generation GTR membranes were primarily non-absorbable membranes like expanded polytetrafluorethylene which required a second surgery for its removal. This led researchers to explore absorbable materials like collagen and synthetic biodegradable polymers to fabricate GTR membranes. In the present study, biodegradable Polylactic acid (PLA) is used to fabricate membranes with the potential to be used for GTR therapy.
Materials and Methods:
Biocompatibility of the PLA membranes were evaluated in a subcutaneous guinea pig model. Antimicrobial effect of the drug-loaded PLA membranes were assessed against a drug-resistant Staphylococcus aureus bacterial isolate. The cytocompatibility of the drug-loaded membranes were evaluated using HeLa cell lines.
The PLA membranes were shown to be biocompatible. The drug-loaded PLA membranes showed significant activity against the bacterial isolate. Among the drug-loaded membranes, tetracycline-loaded membrane showed minimal cellular toxicity.
The results of this study indicate that biodegradable drug-releasing polylactide membranes have the potential to be used for periodontal regeneration. It has the necessary characteristics of a GTR membrane like biocompatibility, space maintaining ability, and tissue integration. Among the various antimicrobial agents loaded in the PLA membranes, tetracycline-loaded membranes exhibited minimal cellular toxicity against HeLa cells; at the same time showing significant activity against a pathogenic bacterium.
Barrier membranes; biocompatibility; cellular toxicity; guided tissue regeneration; polylactic acid; tetracycline loaded
SEARCHGTr is a web-based software for the analysis of glycosyltransferases (GTrs) involved in the biosynthesis of a variety of pharmaceutically important compounds like adriamycin, erythromycin, vancomycin etc. This software has been developed based on a comprehensive analysis of sequence/structural features of 102 GTrs of known specificity from 52 natural product biosynthetic gene clusters. SEARCHGTr is a powerful tool that correlates sequences of GTrs to the chemical structures of their corresponding substrates. This software indicates the donor/acceptor specificity and also identifies putative substrate binding residues. In addition, it provides interfaces to other public databases like GENBANK, SWISS-PROT, CAZY, PDB, PDBSum and PUBMED for extracting various information on GTrs homologous to the query sequence. SEARCHGTr would provide new dimension to our previously developed bioinformatics tool NRPS-PKS. Together, these tools facilitate comprehensive computational analysis of proteins involved in biosynthesis of aglycone core and its downstream glycosylations. Apart from presenting opportunities for rational design of novel natural products, these tools would assist in the identification of biosynthetic products of secondary metabolite gene clusters found in newly sequenced genomes. SEARCHGTr can be accessed at .
The General Time Reversible (GTR) model of nucleotide substitution is at the core of many distance-based and character-based phylogeny inference methods. The procedure described by Waddell and Steel (1997), for estimating distances and instantaneous substitution rate matrices, R, under the GTR model, is known to be inapplicable under some conditions, ie, it leads to the inapplicability of the GTR model. Here, we simulate the evolution of DNA sequences along 12 trees characterized by different combinations of tree length, (non-)homogeneity of the substitution rate matrix R, and sequence length. We then evaluate both the frequency of the GTR model inapplicability for estimating distances and the accuracy of inferred alignments. Our results indicate that, inapplicability of the Waddel and Steel’s procedure can be considered a real practical issue, and illustrate that the probability of this inapplicability is a function of substitution rates and sequence length.
We also discuss the implications of our results on the current implementations of maximum likelihood and Bayesian methods.
GTR model; simulations; nucleotide substitution; homogeneity; phylogeny inference