Synthetic operon for (R,R)-2,3-butanediol production in Bacillus subtilis and Escherichia coli.

To reduce dependence on petroleum, an alternative route to production of the chemical feedstock 2,3-butanediol (2,3-BD) from renewable lignocellulosic sources is desirable. In this communication, the genes encoding the pathway from pyruvate to 2,3-BD (alsS, alsD, and bdhA encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase, respectively) from Bacillus subtilis were engineered into a single tricistronic operon under control of the isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible Pspac promoter in a shuttle plasmid capable of replication and expression in either B. subtilis or Escherichia coli. We describe the construction and performance of a shuttle plasmid carrying the IPTG-inducible synthetic operon alsSDbdhA coding for 2,3-BD pathway capable of (i) expression in two important representative model microorganisms, the gram-positive B. subtilis and the gram-negative E. coli; (ii) increasing 2,3-BD production in B. subtilis; and (iii) successfully introducing the B. subtilis 2,3-BD pathway into E. coli. The synthetic alsSDbdhA operon constructed using B. subtilis native genes not only increased the 2,3-BD production in its native host but also efficiently expressed the pathway in the heterologous organism E. coli. Construction of an efficient shuttle plasmid will allow investigation of 2,3-BD production performance in related organisms with industrial potential for production of bio-based chemicals.

Proposal to rename Carnobacterium inhibens as Carnobacterium inhibens subsp. inhibens subsp. nov. and description of Carnobacterium inhibens subsp. gilichinskyi subsp. nov., a psychrotolerant bacterium isolated from Siberian permafrost.

A novel, psychrotolerant facultative anaerobe, strain WN1359(T), was isolated from a permafrost borehole sample collected at the right bank of the Kolyma River in Siberia, Russia. Gram-positive-staining, non-motile, rod-shaped cells were observed with sizes of 1-2 µm long and 0.4-0.5 µm wide. Growth occurred in the range of pH 5.8-9.0 with optimal growth at pH 7.8-8.6 (pH optimum 8.2). The novel isolate grew at temperatures from 0-37 °C and optimal growth occurred at 25 °C. The novel isolate does not require NaCl; growth was observed between 0 and 8.8 % (1.5 M) NaCl with optimal growth at 0.5 % (w/v) NaCl. The isolate was a catalase-negative, facultatively anaerobic chemo-organoheterotroph that used sugars but not several single amino acids or dipeptides as substrates. The major metabolic end-product was lactic acid in the ratio of 86 % l-lactate : 14 % d-lactate. Strain WN1359(T) was sensitive to ampicillin, chloramphenicol, fusidic acid, lincomycin, monocycline, rifampicin, rifamycin SV, spectinomycin, streptomycin, troleandomycin and vancomycin, and resistant to nalidixic acid and aztreonam. The fatty acid content was predominantly unsaturated (70.2 %), branched-chain unsaturated (11.7 %) and saturated (12.5 %). The DNA G+C content was 35.3 mol% by whole genome sequence analysis. 16S rRNA gene sequence analysis showed 98.7 % sequence identity between strain WN1359(T) and Carnobacterium inhibens. Genome relatedness was computed using both Genome-to-Genome Distance Analysis (GGDA) and Average Nucleotide Identity (ANI), which both strongly supported strain WN1359(T) belonging to the species C. inhibens. On the basis of these results, the permafrost isolate WN1359(T) represents a novel subspecies of C. inhibens, for which the name Carnobacterium inhibens subsp. gilichinskyi subsp. nov. is proposed. The type strain is WN1359(T) ( = ATCC BAA-2557(T) = DSM 27470(T)). The subspecies Carnobacterium inhibens subsp. inhibens subsp. nov. is created automatically. An emended description of C. inhibens is also provided.

The LysR-type transcriptional regulator (LTTR) AlsR indirectly regulates expression of the Bacillus subtilis bdhA gene encoding 2,3-butanediol dehydrogenase.

Bacillus subtilis ferments pyruvate to 2,3-butanediol via α-acetolactate synthase, α-acetolactate decarboxylase, and butanediol dehydrogenase (BDH), encoded by the alsSD operon and the unlinked monocistronic bdhA gene, respectively. Upstream and divergent from alsSD is the alsR gene that encodes AlsR, a member of the LysR-type transcriptional regulator family. AlsR directly stimulates alsSD transcription by binding to characteristic sites preceding the alsS promoter, but its effect on bdhA expression was unknown. The effect of AlsR on bdhA expression was assessed in a wild-type strain and a congenic strain carrying an alsR::spc knockout mutation by measuring: (a) expression of a transcriptional bdhA-lacZ fusion; (b) bdhA mRNA steady-state levels by quantitative reverse transcriptase PCR; and (c) expression of BDH enzymatic activity. Activation of bdhA expression occurred in early stationary phase, and expression was lowered, but not abolished, in the alsR::spc mutant. Mapping the transcriptional start site of bdhA by primer extension revealed a 268-nucleotide 5'-untranslated region preceding the bdhA initiation methionine codon. Transcription initiation was not reduced in the alsR::spc mutant, and by electrophoretic mobility shift assay, purified AlsR protein did not bind to the bdhA promoter region, suggesting that bdhA expression is indirectly under AlsR transcriptional control.

Altered passive eruption diagnosis and treatment: a cone beam computed tomography-based reappraisal of the condition.

AIM: To characterize anatomical features of altered passive eruption (APE)-affected teeth using cone beam computed tomography (CBCT) and to present a novel combined surgical approach to its correction. CLINICAL INNOVATION REPORT: Eighty-four teeth from 14 subjects affected by APE were subjected to CBCT. Periodontal variables were recorded before surgery, and anatomical variables were measured on CBCTs. Clinical crown length was measured on study casts. Surgical treatment was carried out based on the lengths of the anatomical crowns transferred to a surgical guide that served as a reference for the incisions. The mean distance between the CEJ and the bone crest was on average <1 mm, facial bone thickness was ≥ 1 mm and soft tissue thickness was >1 mm for every tooth analysed; no association between the soft and the hard tissue thicknesses was observed. CONCLUSION: The CBCT can be used in the diagnosis and treatment planning of APE cases. Anatomically, the APE cases described often presented a thick facial bone plate.

Antimicrobial photodynamic therapy in the non-surgical treatment of aggressive periodontitis: microbiological profile.

The aim of this trial was to investigate changes occurring in the subgingival microbiological composition of subjects with aggressive periodontitis, treated with antimicrobial photodynamic therapy (aPDT), in a single episode, or scaling and root planing (SRP), in a split-mouth design on -7, 0, and +90 days. Ten patients were randomly assigned to either aPDT using a laser source in conjunction with a photosensitizer or SRP with hand instruments. Subgingival plaque samples were collected and the counts of 40 subgingival species were determined using checkerboard DNA-DNA hybridization. The data were analyzed using the method of generalized estimating equations (GEE) to test the associations between treatments, evaluated parameters, and experimental times (α = .05). The results indicated that aPDT and SRP affects different bacterial species, with aPDT being effective in reducing numbers of A. actinomycetemcomitans than SRP. On the other hand, SRP was more efficient than aPDT in reducing the presence of periodontal pathogens of the Red Complex. Additionally, a recolonization in the sites treated by aPDT was observed, especially for T. forsythia and P. gingivalis. Under our experimental conditions, this trial demonstrates that aPDT and SRP affected different groups of bacteria, suggesting that their association may be beneficial for the non-surgical treatment of aggressive periodontitis.

Bone remodeling adjacent to Morse cone-connection implants with platform switch: a fluorescence study in the dog mandible.

PURPOSE: The aim of this study was to evaluate, through fluorescence analysis, the effect that different interimplant distances, after prosthetic restoration, will have on bone remodeling in submerged and nonsubmerged implants restored with a "platform switch." MATERIALS AND METHODS: Fifty-six Ankylos implants were placed 1.5 mm subcrestally in seven dogs. The implants were placed so that two fixed prostheses, with three interimplant contacts separated by 1-mm, 2-mm, and 3-mm distances, could be fabricated for each side of the mandible. The sides and the positions of the groups were selected randomly. To better evaluate bone remodeling, calcein green was injected 3 days before placement of the prostheses at 12 weeks postimplantation. At 3 days before sacrifice (8 weeks postloading), alizarin red was injected. The amounts of remodeled bone within the different interimplant areas were compared statistically before and after loading in submerged and nonsubmerged implants. RESULTS: Statistically significant differences existed in the percentage of remodeled bone seen in the different regions. Mean percentages of remodeled bone in the submerged and nonsubmerged groups, respectively, were as follows: for the 1-mm distance, 23.0% +/- 0.05% and 23.1% +/- 0.03% preloading and 27.0% +/- 0.03% and 25.2% +/- 0.04% postloading; for the 2-mm distance, 18.2% +/- 0.05% and 18.1% +/- 0.04% preloading and 21.3% +/- 0.07% and 19.9% +/- 0.03% postloading; for the 3-mm distance, 18.3% +/- 0.03% and 18.3% +/- 0.03% preloading and 18.8% +/- 0.04% and 19.8% +/- 0.04% postloading; for distal-extension regions, 16.6% +/- 0.02% and 17.4% +/- 0.04% preloading and 17.0% +/- 0.04% and 18.4% +/- 0.04% postloading. CONCLUSIONS: Based upon this animal study, loading increases bone formation for submerged or nonsubmerged implants, and the interimplant distance of 1 mm appears to result in more pronounced bone remodeling than the 2-mm or 3-mm distances in implants with a "platform switch."

Replacement of hopeless retained primary teeth by immediate dental implants: a case report.

Hopeless retained primary teeth without permanent successors represent a restorative challenge for clinicians, along with esthetic and functional problems for patients. While various treatment approaches for congenitally missing teeth have been proposed, the replacement of a missing tooth with a dental implant offers specific advantages, such as preservation of the alveolar crest and elimination of the need to restore the adjacent teeth, over other options for tooth replacement. The aim of this article was to illustrate the surgical and prosthetic treatment with implants of a patient with primary teeth without permanent successors.

Bacterial and Archaeal Communities Change With Intensity of Vegetation Coverage in Arenized Soils From the Pampa Biome.

Arenization occurs in regions that present sandy soils with normal rainfall levels. Predatory use of environmental sources, the dissolution of arenitic rocks and reworking of non-consolidated surface sands intensify this degradation scenario. Thus, this work aimed to evaluate the impact of the arenization process in the Brazilian Pampa Biome and how this phenomenon affects the soil microbial and plant communities. For this purpose, three arenized areas in Southern Brazil (Pampa Biome) were selected and, in each one, three sampling points were studied: arenized (ARA), arenized to grassland transition (AGT), and grassland (GRA) areas. In the three sampling points, soils presented low levels of nutrients, organic matter, mud and pH acidic in all regions but, the presence of vegetation coverage in AGT and GRA areas preserved the topsoil structure. Our study related ARA with bacterial families Alcaligenaceae, Pseudomonadaceae, and Xanthomonadaceae. AGT with bacterial families Bacillaceae and Burkholderiaceae, and plant species Melinis repens (Willd.) Zizka and Paspalum stellatum Humb. and Bonpl. ex Flüggé, and GRA with bacterial families Koribacteraceae, Hyphomicrobiaceae, and Chthoniobacteraceae, and plant species Croton subpannosus Müll.Arg. ex Griseb., Piptochaetium montevidense (Spreng.) Parodi and Elyonurus sp. The three studied areas (as well as sampling points) present soils extremely poor in nutrients with sandy texture, and the bacterial and plant composition well known to be resistant to environmental stresses were dominant. The vulnerability of these areas causes a degradation scenario, which is worsened by agricultural activities. However, in general, this phenomenon is a natural process that occurs mainly due to soil characteristics (poor soils) and climatic variations.

Optimization in multi-implant placement for immediate loading in edentulous arches using a modified surgical template and prototyping: a case report.

Immediate loading of dental implants shortens the treatment time and makes it possible to give the patient an esthetic appearance throughout the treatment period. Placement of dental implants requires precise planning that accounts for anatomic limitations and restorative goals. Diagnosis can be made with the assistance of computerized tomographic scanning, but transfer of planning to the surgical field is limited. Recently, novel CAD/CAM techniques such as stereolithographic rapid prototyping have been developed to build surgical guides in an attempt to improve precision of implant placement. The aim of this case report was to show a modified surgical template used throughout implant placement as an alternative to a conventional surgical guide.

Antimicrobial photodynamic therapy in the non-surgical treatment of aggressive periodontitis: a preliminary randomized controlled clinical study.

BACKGROUND: The treatment of aggressive periodontitis has always presented a challenge for clinicians, but there are no established protocols and guidelines for the efficient control of the disease. METHODS: Ten patients with a clinical diagnosis of aggressive periodontitis were treated in a split-mouth design study to either photodynamic therapy (PDT) using a laser source with a wavelength of 690 nm associated with a phenothiazine photosensitizer or scaling and root planing (SRP) with hand instruments. Clinical assessment of plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), gingival recession (GR), and relative clinical attachment level (RCAL) were made at baseline and 3 months after treatment with an automated periodontal probe. RESULTS: Initially, the PI was 1.0 +/- 0.5 in both groups. At the 3-month evaluation, the plaque scores were reduced and remained low throughout the study. A significant reduction of GI and BOP occurred in both groups after 3 months (P <0.05). The mean PD decreased in the PDT group from 4.92 +/- 1.61 mm at baseline to 3.49 +/- 0.98 mm after 3 months (P <0.05) and in SRP group from 4.92 +/- 1.14 mm at baseline to 3.98 +/- 1.76 mm after 3 months (P <0.05). The mean RCAL decreased in the PDT group from 9.93 +/- 2.10 mm at baseline to 8.74 +/- 2.12 mm after 3 months (P <0.05), and in the SRP group, from 10.53 +/- 2.30 mm at baseline to 9.01 +/- 3.05 mm after 3 months. CONCLUSION: PDT and SRP showed similar clinical results in the non-surgical treatment of aggressive periodontitis.

The effect of bone condensation and crestal preparation on the bone response to implants designed for immediate loading: a histomorphometric study in dogs.

PURPOSE: The aim of this study was to evaluate the influence of bone condensation and crestal preparation on the bone response of implants designed to promote osseocompression. MATERIALS AND METHODS: In the first phase, the mandibular premolars of 6 dogs were extracted bilaterally. After 8 weeks, each dog received 8 Xive implants (4 per hemimandible). One hemimandible was randomly assigned to the experimental group and the other to the control group. The implant site was prepared using conventional standard drills. Prior to implant placement the crestal drill was used in the experimental group but not in the control group. After 12 weeks, the animals were sedated and sacrificed. The hemimandibles were removed and prepared for histomorphometric analysis of bone-implant contact (BIC) and bone density of areas adjacent to and further from the implant surface. RESULTS: The mean +/- SD percentages of BIC attained were 71.1% +/- 11.8% and 45.1% +/- 16.1% for the experimental and control groups, respectively. The bone density analysis revealed that in the control group, percentage BIC was a mean of 55.6% +/- 11.3% adjacent to the implant and 50.7% +/- 17.9% distant from the implant. In the experimental group, percentage BIC was a mean of 71.1% +/- 8.6% adjacent to the implant and 55.6 +/- 11.3 distant from the implant. The difference between the experimental and control groups was statistically significant for both parameters, BIC and bone density, in the adjacent areas (P < .0001). CONCLUSION: Crestal preparation is of fundamental importance for this implant system, since it led to better bone response, represented by the improved BIC and bone density.

The effects of interimplant distances on papilla formation and crestal resorption in implants with a morse cone connection and a platform switch: a histomorphometric study in dogs.

BACKGROUND: Implant esthetics have been the focus of attention for the last few years, and one of the most important points is the effect that interimplant distances can have on papilla formation and bone loss. The aim of this study was to evaluate the effect that distances of 1, 2, and 3 mm between implants after prosthetic restoration will have on crestal bone resorption (from the top of the implant to the bone crest [TI-BC]) and bone resorption (from the top of the implant to the first bone-to-implant contact TI-BIC) in two-stage implants used in a submerged and non-submerged protocol. METHODS: The mandibular bilateral premolars of seven dogs were extracted, and after 12 weeks, each dog received eight implants. The implants were placed so that three interimplant contact points were created, with 1-mm (group 1), 2-mm (group 2), and 3-mm (group 3) distances constructed on each side. The sides and the position of the groups were randomly selected. After 12 weeks, the implants received metallic prostheses with 5 mm between the contact point and the bone crest. After 8 weeks more, the animals were sacrificed. RESULTS: The TI-BC was 0.20 and 0.18 mm for group 1, 0.15 and 0.14 mm for group 2, and 0.15 and 0.15 mm for group 3 for non-submerged and submerged implants, respectively. At the proximal region, the TI-BC was 0.16 mm for non-submerged and 0.16 mm for submerged implants. The TI-BIC was 0.32 and 0.30 mm for group 1, 0.19 and 0.21 mm for group 2, and 0.30 and 0.24 mm for group 3 for non-submerged and submerged implants, respectively. At the proximal region, the TI-BIC was 0.26 mm for non-submerged and 0.25 mm for submerged implants. There was no statistical difference for any of the parameters (analysis of variance [ANOVA]). CONCLUSION: Distances of 1, 2, and 3 mm between implants do not result in statistically significant differences on TI-BC and TI-BIC around submerged or non-submerged implants with a Morse cone connection and a platform switch.

Influence of interimplant distance on papilla formation and bone resorption: a clinical-radiographic study in dogs.

Implant esthetics has been the focus of attention for the past decade, and one vital issue is the effect of interimplant distance on interimplant papilla formation and crestal bone loss. The aim of this study was to evaluate the effect of 1, 2, and 3 mm of interimplant distance on papilla formation and crestal resorption in submerged and nonsubmerged Ankylos implants after prosthetic restoration. Bilateral mandibular premolars of 7 dogs were extracted, and after 12 weeks each dog received 8 implants. Implants were placed so that 3 interimplant distances were created at 1 mm (group 1), 2 mm (group 2), and 3 mm (group 3). The sides and the position of the groups were randomly selected. Twelve weeks after placement, the implants received metallic prostheses that allowed 5 mm of space between the prosthetic contact point (CP) and the crestal bone (CB). After 8 weeks, the distance between the CP and the papilla (CP-P) and the gingival height at the distal proximal aspect of the prosthesis (CP-DE) was clinically measured. Radiographic images were obtained to measure the distance of the CP to the CB within the interimplant surfaces (CP-IP) and adjacent to the edentulous surfaces (CP-ED). The clinical measurement of CP-P for submerged and nonsubmerged implants was 3.57+/-1.17 mm and 3.10+/-0.82 mm for group 1, 3.57+/-0.78 mm and 3.16+/- 0.87 mm for group 2, and 3.35+/- 0.55 mm and 3.07+/-0.93 mm for group 3. The CP-DE was 3.25+/-0.77 mm for submerged and 2.78+/- 0.64 mm for nonsubmerged implants. The CP-IP for the submerged and nonsubmerged implants was 6.91+/-0.95 mm and 7.68+/-2.73 mm for group 1, 7.46+/-1.43 mm and 5.87+/-1.71 mm for group 2, and 7.72+/-0.81 mm and 7.59+/-1.33 mm for group 3. The CP-ED was 6.77+/-1.33 mm for submerged implants and 6.03+/-1.58 mm for nonsubmerged implants. There were no statistical significant differences for any of the measured parameters. We conclude that when the distance from the CP to the CB was 5 mm, interimplant distances of 1 to 3 mm did not affect papilla formation or crestal resorption of submerged or nonsubmerged implants in the dog model.

Crestal bone loss minimized when following the crestal preparation protocol: a histomorphometric study in dogs.

Initial breakdown of the implant-tissue interface generally begins at the crestal region in successfully osseointegrated implants. The purpose of this study was to evaluate the effect on crestal bone loss (CBL) around implants specially developed for immediate loading with a unique crestal drill. After 8 weeks postextraction, 6 young male mongrel dogs received 48 implants (XiVE) in the region corresponding to the 4 mandibular premolars. The implant sites were prepared according to the manufacturer's protocol with conventional standard drills. Before implant placement, the crestal drill was used in the experimental group but not in the control group. After a healing period of 12 weeks, the dogs were sedated and euthanized. Through linear measurements, from the top of the implant to the first bone-implant contact, the amount of CBL was determined. The histomorphometric results of CBL (mean +/- SEM) were 0.88 +/- 0.13 mm (range 0.0-3.0 mm) in the experimental group and 1.69 +/- 0.17 mm (range 0.0-4.2 mm) in the control group. The difference was statistically significant (P < .05) when the implants were used as the experimental units. The statistical analysis also revealed significance when the dogs were used as the experimental units (P < .05). When the median was used for analyses, the CBL was 0.44 mm for the experimental group and 1.91 mm for the control group. Crestal bone loss was minimized when the crestal preparation protocol was carefully followed by using the osseocondensating XiVE implant system.

Antimicrobial photodynamic therapy for the treatment of teeth with apical periodontitis: a histopathological evaluation.

INTRODUCTION: This study evaluated the in vivo response of apical and periapical tissues of dogs' teeth with apical periodontitis after one-session endodontic treatment with and without antimicrobial photodynamic therapy (aPDT). METHODS: Sixty root canals with experimentally induced apical periodontitis were instrumented and assigned to 4 groups receiving aPDT and root canal filling (RCF) or not: group aPDT+/RCF+ (n = 20): aPDT (photosensitizer phenothiazine chloride at 10 mg/mL for 3 minutes and diode laser [λ = 660 nm, 60 mW/cm(2)] for 1 minute) and RCF in the same session; group aPDT+/RCF- (n = 10); group aPDT-/RCF+ (n = 20), and group aPDT-/RCF- (n = 10). Teeth were restored, and the animals were killed after 90 days. Sections from the maxillas and mandibles were stained with hematoxylin-eosin and Mallory trichrome and examined under light microscopy. Descriptive (ie, newly formed apical mineralized tissue, periapical inflammatory infiltrate, apical periodontal ligament thickness, and mineralized tissue resorption) and quantitative (ie, periapical lesion size and number of inflammatory cells) microscopic analysis was performed. Quantitative data were analyzed by the Kruskal-Wallis and Dunn tests (α = .05). RESULTS: In the aPDT-treated groups, the periapical region was moderately/severely enlarged with no inflammatory cells, moderate neoangiogenesis and fibrogenesis, and the smallest periapical lesions. CONCLUSIONS: Although apical closure by mineralized tissue deposition was not achieved, the absence of inflammatory cells, moderate neoangiogenesis, and fibrogenesis in the periapical region in the groups treated with aPDT indicate that this can be a promising adjunct therapy to cleaning and shaping procedures in teeth with apical periodontitis undergoing one-session endodontic treatment.