Ectoine hyperproduction by engineered Halomonas bluephagenesis.

Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine.

Elucidating the salt-tolerant mechanism of Halomonas cupida J9 and unsterile ectoine production from lignocellulosic biomass.

BACKGROUND: Ectoine as an amino acid derivative is widely applied in many fields, such as the food industry, cosmetic manufacturing, biologics, and therapeutic agent. Large-scale production of ectoine is mainly restricted by the cost of fermentation substrates (e.g., carbon sources) and sterilization. RESULTS: In this study, Halomonas cupida J9 was shown to be capable of synthesizing ectoine using xylose as the sole carbon source. A pathway was proposed in H. cupida J9 that synergistically utilizes both WBG xylose metabolism and EMP glucose metabolism for the synthesis of ectoine. Transcriptome analysis indicated that expression of ectoine biosynthesis module was enhanced under salt stress. Ectoine production by H. cupida J9 was enhanced by improving the expression of ectoine biosynthesis module, increasing the intracellular supply of the precursor oxaloacetate, and utilizing urea as the nitrogen source. The constructed J9U-P8EC achieved a record ectoine production of 4.12 g/L after 60 h of xylose fermentation. Finally, unsterile production of ectoine by J9U-P8EC from either a glucose-xylose mixture or corn straw hydrolysate was demonstrated, with an output of 8.55 g/L and 1.30 g/L of ectoine, respectively. CONCLUSIONS: This study created a promising H. cupida J9-based cell factory for low-cost production of ectoine. Our results highlight the potential of J9U-P8EC to utilize lignocellulose-rich agriculture waste for open production of ectoine.

Effect of cDMEM media containing Ectoine on human periodontal ligament mesenchymal stem cell survival and differentiation.

BACKGROUND/AIM: Ectoine is an amino acid that can preserve osmotic balance and has more preservative activity than other osmoregulators. There are no published reports on the osmoregulators' effects on viability or differentiation of dental stem cells. The aim of this study was to investigate the effect of Ectoine as a storage media on the viability and differentiation potential of human periodontal ligament mesenchymal stem cells (hPDLMSCs). MATERIALS AND METHODS: hPDLMSCs were obtained from impacted third molar teeth. The cells were isolated, submitted to trilineage differentiation, and characterized by flow cytometer (FC). hPDLMSCs were incubated with different media containing Ectoine, complete DMEM (cDMEM), Ectoine+cDMEM, milk, and tap water for 2, 6, 12, and 24 h. The cells were analyzed by FC for viability, early-apoptosis, late apoptosis, and necrosis rates. Osteogenic and fibroblastic differentiation in hPDLMSCs were investigated by Alizarin red stain and vimentin expression. RESULTS: All treated groups showed significant decreases in cell viability after 2 h. Significant increases were detected in the number of dead cells between 2 and 12 h in all groups except the Ectoine+cDMEM group. The deposition of mineral matrix nodules was significantly higher in cells cultured with Ectoine+cDMEM compared with the other media. Higher vimentin expressions were detected in cells cultured with cDMEM and Ectoine+cDMEM media, respectively. CONCLUSIONS: Ectoine added to cDMEM media, promoted cell survival plus osteogenic and fibroblastic differentiation of hPDLMSCs.

Efficacy and tolerability of an ectoine mouth and throat spray compared with those of saline lozenges in the treatment of acute pharyngitis and/or laryngitis: a prospective, controlled, observational clinical trial.

The aim of this observational trial was to evaluate the efficacy and tolerability of a mouth and throat spray containing ectoine in the treatment of acute pharyngitis and/or laryngitis. The outcome was compared with control treatment using saline lozenges. This study was designed as a prospective, controlled, non-randomized, observational multicenter clinical trial and was conducted in Germany. The study population consisted of 95 patients. The decision for treatment with either spray or lozenges was based on the patients' preference for pharyngeal or oral application. Investigators assessed symptoms specific to acute pharyngitis/laryngitis and determined the pharyngitis symptom score. Both patients and investigators evaluated the tolerability and efficacy of the treatment applied. Treatment with the spray showed higher efficacy, 1.95 ± 0.81 versus 1.68 ± 0.67 (investigators) and 1.97 ± 0.88 versus 1.57 ± 0.69 (patients, p < 0.05). Treatment with the spray resulted in significantly greater reduction of cervical lymph node swelling (p < 0.05), ∆ spray = 0.44 ± 0.62, ∆ lozenges = 0.21 ± 0.62. The lozenges showed some advantage in relieving cough, ∆ lozenges = 0.62 ± 0.94 versus ∆ spray = 0.44 ± 0.85. Both patients and investigators rated the tolerability of both medical devices as "good" to "very good". Adverse events of mild to moderate severity were either possibly related or not related to the medical devices used. No serious adverse events occurred. Taken together, while the tolerability was consistent in both treatment groups, the ectoine-based spray showed superior efficacy in treating acute pharyngitis and/or laryngitis.

[Aerobic methylobacteria as promising objects of modern biotechnology].

The experimental data of the past decade concerning the metabolic peculiarities of aerobic meth ylobacteria and the prospects for their use in different fields of modern biotechnology, including genetic engineering techniques, have been summarized.

[Biosynthesis of secondary metabolites in methanotrophs: biochemical and genetic aspects (review)].

The review summarizes the data on the metabolic potential of methanotrophs as producers of biopolymers, alternative biofuel, bioprotectants, and other secondary metabolites. The work provides the examples of modern 'omic' technologies used for genetic engineering of efficient methanotrophic producers.

Green electrochemical and chromatographic quantifications of the extremolyte ectoine in halophilic bacterial cultures and related pharmaceutical preparations.

The realistic implementation of electrochemistry into bacterial biosynthesis monitoring programs has always been a challenging task. In this study, two simple, rapid, selective, and sensitive potentiometric sensors were developed and optimized for quantitative analysis of the extremolyte / osmoprotector ectoine (ECT) in halophilic bacterial cultures and also in its related pharmaceutical products. The developed sensors were a Polyvinyl chloride membrane sensor (ECT-PVC) and a coated graphite sensor (ECT-CG). They were established based on the ion association complex of ectoine cation with phosphotungstic acid (ECT-PTA) counter anion as ion exchange site using dioctyl-phthalate (DOP) as a solvent mediator or plasticizer. The sensors exhibited fast, stable, selective, and linear Nernstian responses over a broad range of concentrations ranging from 1 × 10-5 to 1 × 10-2 M of the studied drug. The developed sensors were optimized and cross-validated with a proposed HPLC method according to ICH guidelines. The proposed methods were successfully employed to quantify the studied drug in three different types of halophilic bacterial cultures and in an ectoine nasal spray pharmaceutical product. The selected bacteria species were Chromohalobacter salexigens, Halobacillus halophilus, and Halomonas elongata. The proposed methods were statistically compared with the reported methods, demonstrating no significant difference with respect to accuracy and precision. Greenness and environmental impact were also evaluated for the proposed procedures, verifying that they were excellent green and eco-friendly analytical methods.

What Does Ectoine Do to DNA? A Molecular-Scale Picture of Compatible Solute-Biopolymer Interactions.

Compatible solutes accumulate in the cytoplasm of halophilic microorganisms, enabling their survival in a high-salinity environment. Ectoine is such a compatible solute. It is a zwitterionic molecule that strongly interacts with surrounding water molecules and changes the dynamics of the local hydration shell. Ectoine interacts with biomolecules such as lipids, proteins, and DNA. The molecular interaction between ectoine and biomolecules, in particular the interaction between ectoine and DNA, is far from being understood. In this paper, we describe molecular aspects of the interaction between ectoine and double-stranded DNA (dsDNA). Two 20 base pairs-long dsDNA fragments were immobilized on a gold surface via a thiol-tether. The interaction between the dsDNA monolayers with diluted and concentrated ectoine solutions was examined by means of X-ray photoelectron and polarization modulation infrared reflection absorption spectroscopies (PM IRRAS). Experimental results indicate that the ability of ectoine to bind water reduces the strength of hydrogen bonds formed to the ribose-phosphate backbone in the dsDNA. In diluted (0.1 M) ectoine solution, DNA interacts predominantly with water molecules. The sugar-phosphate backbone is involved in the formation of strong hydrogen bonds to water, which, over time, leads to a reorientation of the planes of nucleic acid bases. This reorientation destabilizes the strength of hydrogen bonds between the bases and leads to a partial dehybridization of the dsDNA. In concentrated ectoine solution (2.5 M), almost all water molecules interact with ectoine. Under this condition, ectoine is able to interact directly with DNA. Density functional theory (DFT) calculations demonstrate that the direct interaction involves the nitrogen atoms in ectoine and phosphate groups in the DNA molecule. The results of the quantum-chemical calculations show that rearrangements in the ribose-phosphate backbone, caused by a direct interaction with ectoine, facilitates contacts between the O atom in the phosphate group and H atoms in a nucleic acid base. In the PM IRRA spectra, an increase in the number of IR absorption modes in the base pair frequency region proves that the hydrogen bonds between bases become weaker. Thus, a sequence of reorientations caused by interaction with ectoine leads to a breakdown of hydrogen bonds between bases in the double helix.

Rational flux-tuning of Halomonas bluephagenesis for co-production of bioplastic PHB and ectoine.

Ectoine, a compatible solute synthesized by many halophiles for hypersalinity resistance, has been successfully produced by metabolically engineered Halomonas bluephagenesis, which is a bioplastic poly(3-hydroxybutyrate) producer allowing open unsterile and continuous conditions. Here we report a de novo synthesis pathway for ectoine constructed into the chromosome of H. bluephagenesis utilizing two inducible systems, which serve to fine-tune the transcription levels of three clusters related to ectoine synthesis, including ectABC, lysC and asd based on a GFP-mediated transcriptional tuning approach. Combined with bypasses deletion, the resulting recombinant H. bluephagenesis TD-ADEL-58 is able to produce 28 g L-1 ectoine during a 28 h fed-batch growth process. Co-production of ectoine and PHB is achieved to 8 g L-1 ectoine and 32 g L-1 dry cell mass containing 75% PHB after a 44 h growth. H. bluephagenesis demonstrates to be a suitable co-production chassis for polyhydroxyalkanoates and non-polymer chemicals such as ectoine.

Application of a cell sheet-polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability.

We have succeeded in fabricating a cell sheet-polymer film complex involving a temperature-sensitive polymer that has enough mechanical strength that can be manipulated even by forceps. The polymer film can be removed by lowering the temperature after transplantation, demonstrating its potential use in regenerative medicine. Recently, tissue engineering involving cell sheets was developed, tissues being fabricated by layering of these cell sheets. This technique promises high density cell packing, which is important for native cell functions, and successful heart therapy using cardiac cell sheets has been reported. On the other hand, the fabrication of a large tissue using cell sheets is difficult because of fragility of the cell sheets. Here, we have developed a novel method in which cells are attached to a temperature-sensitive poly-N-isopropylacrylamide film mixed with laminin and collagen IV, and report that the cell sheet-polymer film complex can be manipulated with forceps. A cell sheet can be removed from the polymer film by lowering the temperature after the manipulation. We have utilized this technique for the primary myocardium and fabricated a physiologically active multi-layered cardiac cell sheet. By applying a micropattern to this polymer film, we have succeeded in making a skeletal muscle cell sheet in which myotubes are oriented in the desired direction. Overall, we showed that this method is useful for cell sheet manipulation, morphogenesis, and transplantation.

A process for the production of ectoine and poly(3-hydroxybutyrate) by Halomonas boliviensis.

The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L(-1)) at 10-15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L(-1)) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L(-1), respectively, were obtained at 5-7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed-the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH (4) (+) , and PO (4) (3-) . In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L(-1) h(-1) and ectoine concentration, content, and volumetric productivity of 4.3 g L(-1), 7.2 wt.%, and 2.8 g L(-1) day(-1), respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L(-1) day(-1).

Effect of ectoine, hydroxyectoine and ß-hydroxybutyrate on the temperature and pressure stability of phospholipid bilayer membranes of different complexity.

Previous research has shown that ectoines fluidize lipid monolayers by increasing the liquid expanded region in DPPC monolayers and also decreasing the line tension responsible for the phase morphology. Here, we explored possible effects of the compatible osmolytes ectoine, hydroxyectoine and ß-hydroxybutyrate on lipid bilayer membranes, including effects of temperature and pressure. The effect of the protective osmolytes on the phase transition of DPPC bilayers was investigated by fluorescence spectroscopy, differential scanning calorimetry and pressure perturbation calorimetry. A slight change of the phase behavior was observed, which resulted in a stabilization of the gel phase, which may be caused by an alteration of the hydration properties at the lipid interface and H-bond and electrostatic interactions in the headgroup region. We then explored the cosolvents' effects on giant unilamellar vesicles (GUVs) formed by lipid mixtures exhibiting phase separation into liquid-ordered (lo) and liquid-disordered (ld) domains using BODIPY-PC and the DiI18 dye as labels. The presence of both, ectoine and hydroxyectoine showed significant effects on the lateral organization increasing the fluid domains. Moreover, we observed a considerable increase in the adhesion behavior of small vesicles onto GUV surfaces. Diffusion studies by fluorescence recovery after photobleaching experiments on POPC giant vesicles quantitatively showed a hydroxyectoine-induced increase of the diffusion coefficient values, clearly demonstrating an increase in the lateral mobility of lipid within the bilayer membrane. This study provides clear evidence for the fluidizing effect of the compatible solutes on bilayer lipid membranes. A marked effect, however, was only detected if phase separated domains exist.

Abscisic acid promotes accumulation of toxin ODAP in relation to free spermine level in grass pea seedlings (Lathyrus sativus L.).

Interrelationship among abscisic acid (ABA) content, accumulation of free polyamines and biosynthesis of beta-N-oxalyl-l-alpha,beta-diaminopropionic acid (ODAP) was studied in grass pea (Lathyrus sativus L.) seedlings under drought stress induced by 10% polyethylene glycol (PEG6000). Increase of ABA content occurred prior to that of ODAP and polyamine contents, and was found significantly positive correlation between ABA content and ODAP content. Addition of exogenous ABA increased ODAP content in leaves. On the other hand, pretreatment with alpha-difluoromethylarginine (DFMA), a polyamine biosynthesis inhibitor, significantly suppressed the accumulation of free putrescine (Put), free spermidine (Spd) and free spermine (Spm), which in turn inhibited biosynthesis of ODAP in well-watered leaves. Meanwhile, addition of exogenous Put alleviated DFMA-induced inhibition on the biosynthesis of Put and Spd, but did not affect the biosynthesis of Spm and ODAP in well-watered leaves. Same result was also achieved in drought-stressed leaves. Increasing accumulation of ODAP was significantly correlated with increasing Spm content (R=0.7957**) but not with that of Spd and Put. Therefore, it can be argued that ABA stimulated the biosynthesis of ODAP simultaneously with increasing the level of free Spm under drought stress condition.

Water-compatible molecularly imprinted polymers for selective solid phase extraction of dencichine from the aqueous extract of Panax notoginseng.

Specific molecularly imprinted polymers for dencichine were developed for the first time in this study by the bulk polymerization using phenylpyruvic acid and dl-tyrosine as multi-templates. The photographs confirmed that molecularly imprinted polymers prepared using N,N'-methylene diacrylamide as cross-linker and glycol dimethyl ether as porogen displayed excellent hydrophilicity. Selectivity, adsorption isotherm and adsorption kinetics were investigated. The sample loading-washing-eluting solvent was optimized to evaluate the property of molecularly imprinted solid phase extract. Compared with LC/WCX-SPE, water-compatible molecularly imprinted solid phase extraction displayed more excellent specific adsorption performance. The extracted dencichine from Panax notoginseng with the purity of 98.5% and the average recovery of 85.6% (n=3) was obtained.

[Biotechnological potential of methylotrophic bacteria: a review of current status and future prospects].

Major results of the authors' findings on the implementation of biotechnological potential of aerobic methylobacteria and methanotrophs for obtaining forage proteins, biopolymers (polybutyrate and polysaccharides), enzymes (oxidoreductases), and bioprotectors (ectoin), as well as for degrading toxic C1 and Cn compounds have been reviewed. Unique features of the structural and functional organization of the metabolism of extremophilic (tolerant) methylotrophs are discussed, with a view for their prospective use in various fields of modern biotechnology, including biocatalysis and nanotechnology.

Water stress and accumulation of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid in grass pea (Lathyrus sativus).

Grass pea seedlings were grown in an irrigated field. Roots of 15-day-old seedlings were treated with PEG, and leaves were studied. With the duration of PEG treatment, changes in the lipid peroxidation and activities of superoxide dismutase, catalase, peroxidase, and glutathione reductase as well as contents of hydrogen peroxide and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP) were assayed. The results indicate that with the duration of PEG treatment, activities of superoxide dismutase, peroxidase, and catalase decreased, whereas contents of hydrogen peroxide and ODAP, extent of lipid peroxidation, and activity of glutathione reductase increased. Both diethyldithiocarbamate and aminotriazole strongly inhibit activities of superoxide dismutase and catalase, respectively. At same time, the extent of lipid peroxidation was obviously increased. However, mannitol decreased the extent of lipid peroxidation. Diethyldithiocarbamate, aminotriazole, and mannitol do not affect the accumulation of ODAP. The observations suggest that there is no direct relationship between the accumulation of ODAP and the metabolism of free radicals. In addition, the relationship between water stress and ODAP accumulation in grass pea is discussed.

The thermal polymerization of amino acids in the presence of sand.

Sand was tested as a model of a common "impurity" that could have influenced the formation of thermal prebiotic protein. Increasing proportions of sand (0-16 g) in admixture with one set of reactant amino acids (1g), when heated at 175 degrees C for 1.5 h, resulted in increasing yields of polyamino acids of increasing size and color intensity; amino acid composition was not greatly affected. Similar results were noted for three of five other sets of reactant amino acids (8 g sand per g amino acids). In no case did sand prevent the amino acids from polymerizing. The results are interpreted to indicate a broader range of conditions conducive to the formation of prebiotic protein and to further emphasize that environmental parameters should be considerided in the experimental modeling of prebiotic processes.

Ectoine production from lignocellulosic biomass-derived sugars by engineered Halomonas elongata.

In this study, the water-retaining cyclic amino acid ectoine was produced from a variety of sugars, including glucose, xylose, cellobiose, and glucose/xylose mixture using engineered Halomonas elongata. When grown on xylose as the sole carbon source, H. elongata produced 333 mmol/kg fresh cell weight (FW) of ectoine, which was 1.4-fold higher than that produced from glucose. To improve ectoine production, an ectD deficient H. elongata mutant was constructed. The engineered H. elongata produced 377 mmol/kg FW of ectoine from a glucose/xylose mixture. Ectoine was also produced from rice straw hydrolysate. These results show that H. elongata can produce ectoine from a variety of sugars derived from lignocellulosic biomass and thus has tremendous potential as a host for producing useful compounds from biomass resources.

Eficácia do diamino fluoreto de prata em paralisar lesões de cárie em dentina: ensaios laboratorial e clínico controlado e randomizado / Efficacy of silver diamine fluoride in arresting dentin caries lesions: controlled randomized clinical tria

Este estudo objetivou: (1) investigar a eficácia in vitro do diamino fluoreto de prata (DFP) em paralisar lesões de cárie em dentina após diferentes concentrações e tempos de aplicação; (2) comparar o impacto do DFP e do tratamento restaurador atraumático (TRA) na qualidade de vida relacionada à saúde bucal (QVRSB) de préescolares; e (3) avaliar a eficácia do DFP comparado ao TRA, em paralisar lesões de cárie, por meio de um ensaio clínico controlado e randomizado após 2 anos de acompanhamento. Para o objetivo 1, blocos de dentina (n=42) foram fixados em placas de poliestireno. Um inóculo bacteriano misto (1,5x108 UFC/mL) foi adicionado ao meio de cultura com sacarose 5%, contido nas placas, que foram incubadas para a formação de biofilme. As amostras foram escaneadas em micro CT (M1) e tratadas com DFP de acordo com os grupos (n=6): DFP 30%, aplicação imediata; DFP 30%, 1'; DFP 30%, 3'; DFP 38%, aplicação imediata; DFP 38%, 1'; DFP 38%, 3'. Um grupo controle, sem tratamento, também foi preparado. Após o escaneamento (M2), os blocos foram submetidos a um desafio cariogênico (21 dias) e novamente escaneados (M3). O pH do meio de cultura e a profundidade das lesões inter e intra grupos foram comparados pelos testes de Kruskal-Wallis e Wilcoxon. Para os objetivos 2 e 3, préescolares com ao menos uma lesão de cárie ativa na oclusal de seus molares decíduos foram randomicamente alocados em dois grupos: DFP e TRA. Os índicesceo-d/CPO-DICDAS foram usados para detecção da presença e atividade de cárie. O B-ECOHIS foi usado para avaliar a QVRSB antes (M1), 15 dias (M2) e 3 meses (M3) após os tratamentos. O sucesso clínico foi avaliado após 6, 12, 18 e 24 meses. Foram ainda avaliados: tempo do procedimento, efeitos adversos/percepção estética e ansiedade. No estudo in vitro, em M1 e M2, não houve diferença na profundidade das lesões entre os grupos (p>0,05). Em M3, o DFP 38% apresentou menor pH do biofilme e os grupos que receberam aplicação por 1' e 3', em ambas as concentrações, não apresentaram aumento na profundidade da lesão em relação ao M1. DFP e TRA não diferiram quanto ao B-ECOHIS total, CIS e FIS em M2 e M3 e o B-ECOHIS total diminuiu de M1 para M2 e M3 em ambos os grupos (p<0,05). Após 2 anos, não houve diferença entre os tratamentos quanto à paralisação da cárie (p=0,072) e o tempo de tratamento para o DFP foi menor (p<0,001). Não houve diferença entre os grupos quanto aos efeitos adversos/percepção estética observados pelo operador (p=0,816) e pelos pais (p=1,000). A ansiedade não mudou após os tratamentos (p=0,583). Assim, o tempo mínimo de aplicação do DFP 30% para paralisar lesões de cárie foi de 1', enquanto o 38% paralisou com aplicação imediata, in vitro. Tanto o DFP quanto TRA melhoraram a QVRSB dos pré-escolares, sem diferença entre eles. Ainda, o DFP mostrou-se semelhante ao TRA na paralisação de cárie, ansiedade e efeitos adversos, requerendo menor tempo de cadeira. (AU)

This study aimed to: (1) investigate the in vitro efficacy of silver diamine fluoride (SDF) in arresting dentin caries lesions after different concentrations and application times; (2) compare the impact of SDF and atraumatic restorative treatment (ART) on the oral health-related quality of life (QHRQoL) in preschoolers; and (3) to evaluate the effectiveness of SDF compared to ART, in arresting caries lesions, through a controlled randomized clinical trial after 2 years of follow-up. For objective 1, dentin blocks (n=42) were fixed in polystyrene plates. A mixed bacterial inoculum (1.5x108 CFU/mL) was added to the culture medium with 5% sucrose, in the plates; that were incubated for biofilm formation. The samples were scanned on micro CT (M1) and treated with SDF according to the groups (n=6): SDF 30%, immediate application; SDF 30%, 1'; SDF 30%, 3'; SDF 38%, immediate application; SDF 38%, 1'; SDF 38%, 3'. A control group, without treatment, was also prepared. After scanning (M2), the blocks were submitted to a cariogenic challenge (21 days) and scanned again (M3). The pH of the culture medium and the depth of lesions between and within the groups were compared using the Kruskal-Wallis and Wilcoxon tests. For objectives 2 and 3, preschoolers with at least one active caries lesion on the occlusal surface of their primary molars were randomly allocated into two groups: SDF and ART. The indexes dmft/DMFT and ICDAS were used to detect the presence and activity of caries. B-ECOHIS was used to assess the QHRQoL of children before (M1), 15 days (M2) and 3 months (M3) after treatments. Clinical success was assessed after 6, 12, 18 and 24 months. Were also evaluated: time of the procedure, adverse effects/aesthetic perception and the child's anxiety. In the in vitro study, in M1 and M2, there was no difference in the depth of the lesions between the groups (p>0.05). In M3, SDF 38% had a lower pH of the biofilm and the groups that received application for 1' and 3', in both concentrations, did not present an increase in the depth of the lesion in relation to M1. SDF and ART did not differ in total B-ECOHIS, CIS and FIS in M2 and M3; and the total B-ECOHIS decreased from M1 to M2 and M3 for both groups (p<0.05). After 2 years, there was no difference between treatments regarding caries arrest (p=0.072) and the treatment time for SDF was shorter (p<0.001). There was no difference regarding the adverse effects/aesthetic perception observed by the operator (p=0.816) and reported by the parents (p=1.000), according to the groups. Anxiety did not change either before or after treatments (p=0.583). Thus, the minimum time of application of SDF 30% to arrest dentin caries was 1', while SDF 38% arrested with immediate application, in vitro. Both SDF and ART improved the QHRQoL of preschoolers, with no difference between them. Still, SDF was similar to ART in arresting caries, anxiety and adverse effects, requiring less chair time. (AU)

Tolerance of plastic-encapsulated Pseudomonas putida KT2440 to chemical stress.

Pseudomonas putida dried in the presence of hydroxyectoine or trehalose can withstand exposure to organic solvents and therefore can be encapsulated inside plastics such as polystyrene. Here we show that P. putida in a plastic-encapsulated dried tablet exhibits remarkable tolerance to chemical stress, comparable to that of spores of Bacillus subtilis.