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This study aimed to evaluate the disruptive effect of fungal mutanase against cariogenic biofilm after short-term treatment. For that, mature Streptococcus mutans biofilms (n = 9) were exposed to active or inactivated enzymes produced by Trichoderma harzianum for 1 min, two times per day. Biofilms were analyzed by amount of matrix water-insoluble polysaccharides, bacterial viability, acidogenicity, and morphology by scanning electron microscopy (SEM). The group treated with active enzymes (AE) had a significantly lower amount of insoluble polysaccharides (893.30 ± 293.69) when compared to the negative control group (NaCl, 2192.59 ± 361.96), yet no significant difference was found when comparing to the positive control group (CHX, 436.82 ± 151.07). Also, there was no significant effect on bacteria metabolism and viability (P-value < 0.05). Data generated by the quantitative analysis were confirmed through scanning electron microscopy images. Thus, fungal mutanase degraded the biofilm after a short-term treatment without interfering with bacterial viability and metabolism. Such findings offer insight to the development of routine oral care products containing this input.

Topical ophthalmic antibiotics show low efficacy due to the well-known physiological defense mechanisms of the eye, which prevents the penetration of exogenous substances. Here, we aimed to incorporate besifloxacin into liposomes containing amines as positively charged additives and to evaluate the influence of this charge on drug delivery in two situations: (i) iontophoretic and (ii) passive treatments. Hypothesis are (i) charge might enhance the electromigration component upon current application improving penetration efficiency for a burst drug delivery, and (ii) positive charge might prolong formulation residence time, hence drug penetration. Liposomes elaborated with phosphatidylcholine (LP PC) or phosphatidylcholine and spermine (LP PC: SPM) were stable under storage at 6 ºC for 30 days, showed mucoadhesive characteristics, and were non-irritant, according to HET-CAM tests. Electron paramagnetic resonance spectroscopy measurements showed that neither the drug nor spermine incorporations produced evident alterations in the fluidity of the liposome's membranes, which retained their structural stability even under iontophoretic conditions. Mean diameter and zeta potential were 177.2 ± 2.7 nm and − 5.7 ± 0.3 mV, respectively, for LP PC; and 175.4 ± 1.9 nm and + 19.5 ± 1.0 mV, respectively, for LP PC:SPM. The minimal inhibitory concentration (MIC) and the minimal bactericide concentration (MBC) of the liposomes for P. aeruginosa showed values lower than the commercial formulation (Besivance). Nevertheless, both formulations presented a similar increase in permeability upon the electric current application. Hence, liposome charge incorporation did not prove to be additionally advantageous for iontophoretic therapy. Passive drug penetration was evaluated through a novel in vitro ocular model that simulates the lacrimal flow and challenges the formulation resistance in the passive delivery situation. As expected, LP PC: SPM showed higher permeation than the control (Besivance). In conclusion, besifloxacin incorporation into positively charged liposomes improved passive topical delivery and can be a good strategy to improve topical ophthalmic treatments.

Objectives The aim of the present clinical randomized split-mouth study was to evaluate the effectiveness and efficiency of an Er:YAG laser for caries removal in primary molars, microbiological dentin analysis, and clinical restorations after 1 year in 29 children. Materials and methods The children’s teeth were randomized into two groups: (I) an Er:YAG laser group and (II) a bur preparation group. The efficiency of the treatments (the time necessary for the removal of carious tissue) was evaluated based on the time spent on caries removal in the deciduous molars. The effectiveness (caries removal capacity) of the caries removal was determined by means of a blind test in which the examiner performed a tactile and visual examination of the dentin. Microbiological analysis was performed by counting the Streptococcus mutans and Lactobacillus sp in the remaining dentin. Clinical analysis of restorations was performed using the USPHS method in combination with photographs of restored teeth, 7 days after the restorative procedure and again after 1 year. All cavities were restored with the Adper Single Bond 2/Filtek Z350 system. The obtained data were analyzed with a significance level of 5 %. Results The Er:YAG laser was less effective and had the same efficacy as bur preparation during caries removal at the pulpal wall of deciduous molars. In the surrounding walls, bur preparation was the more effective method. Regardless of the method employed, the affected dentin in the pulpal wall had similar amounts of S. mutans and Lactobacillus sp . The restorations were clinically accepted by the USPHS method over a 1-year period. Conclusion It can be concluded that caries removal with an Er:YAG laser has no influence on the clinical behavior of restorations. Clinical relevance Irradiation with an Er:YAG laser is appropriate for caries removal in primary teeth.

Introduction: This study evaluated the impact of CO2 laser treatment and acidulated phosphate fluoride (APF) on enamel demineralization and biofilm formation, using in vitro and in situ designs. Methods: Demineralized enamel slabs were distributed among 8 groups: placebo, placebo + continuous CO2 laser, placebo + repeated CO2 laser, placebo + ultrapulsed CO2 laser, 1.23% APF, APF + continuous CO2 laser, APF + repeated CO2 laser and APF + ultrapulsed CO2 laser. In the in vitro study, 15 enamel slabs from each group were subjected to a pH-cycling regimen for 14 days. In the cross over in situ design, 11 volunteers wore palatal appliances with demineralized enamel slabs for 2 periods of 14 days each. Drops of sucrose solution were dripped onto enamel slabs 8×/day. Biofilms formed on slabs were collected and the colony-forming units (CFU) of Streptococcus mutans and Lactobacillus were determined. Results: For both in vitro and in situ studies, there was no significant difference between treatments (P>0.05). However, all treatments increased microhardness of demineralized enamel (P<0.05). After a further in situ cariogenic challenge, with the exception of the placebo, all treatments maintained microhardness values (P<0.05). Microbiological analysis showed no difference in Streptococcus mutans (P>0.05) or Lactobacillus (P>0.05) counts between groups. Conclusion: The results suggest that APF gel combined with the CO2 laser, regardless of the pulse emission mode used, was effective in controlling enamel demineralization, but none of the tested treatments was able to prevent bacterial colonization.

Introduction: This study evaluated the impact of CO2 laser treatment and acidulated phosphate fluoride (APF) on enamel demineralization and biofilm formation, using in vitro and in situ designs. Methods: Demineralized enamel slabs were distributed among 8 groups: placebo, placebo + continuous CO2 laser, placebo + repeated CO2 laser, placebo + ultrapulsed CO2 laser, 1.23% APF, APF + continuous CO2 laser, APF + repeated CO2 laser and APF + ultrapulsed CO2 laser. In the in vitro study, 15 enamel slabs from each group were subjected to a pH-cycling regimen for 14 days. In the cross over in situ design, 11 volunteers wore palatal appliances with demineralized enamel slabs for 2 periods of 14 days each. Drops of sucrose solution were dripped onto enamel slabs 8×/day. Biofilms formed on slabs were collected and the colony-forming units (CFU) of Streptococcus mutans and Lactobacillus were determined. Results: For both in vitro and in situ studies, there was no significant difference between treatments (P > 0.05). However, all treatments increased microhardness of demineralized enamel (P < 0.05). After a further in situ cariogenic challenge, with the exception of the placebo, all treatments maintained microhardness values (P < 0.05). Microbiological analysis showed no difference in Streptococcus mutans (P > 0.05) or Lactobacillus (P > 0.05) counts between groups. Conclusion: The results suggest that APF gel combined with the CO2 laser, regardless of the pulse emission mode used, was effective in controlling enamel demineralization, but none of the tested treatments was able to prevent bacterial colonization.

Brewer’s spent grain (BSG) is the largest by-product originated from the brewery industry with a high potential for producing carbohydrases by solid-state fermentation. This work aimed to test the efficacy of a carbohydrases-rich extract produced from solid-state fermentation of BSG, to enhance the digestibility of a plant-based diet for European seabass ( Dicentrarchus labrax ). First, BSG was fermented with A. ibericus to obtain an aqueous lyophilized extract (SSF-BSG extract) and incorporated in a plant-based diet at increasing levels (0—control; 0.1%, 0.2%, and 0.4%). Another diet incorporating a commercial carbohydrases-complex (0.04%; Natugrain; BASF) was formulated. Then, all diets were tested in in vitro and in vivo digestibility assays. In vitro assays, simulating stomach and intestine digestion in European seabass, assessed dietary phosphorus, phytate phosphorus, carbohydrates, and protein hydrolysis, as well as interactive effects between fish enzymes and dietary SSF-BSG extract. After, an in vivo assay was carried out with European seabass juveniles fed selected diets (0—control; 0.1%, and 0.4%). In vitro digestibility assays showed that pentoses release increased 45% with 0.4% SSF-BSG extract and 25% with Natugrain supplemented diets, while amino acids release was not affected. A negative interaction between endogenous fish enzymes and SSF-BSG extract was observed in both diets. The in vivo digestibility assay corroborated in vitro data. Accordingly, the dietary supplementation with 0.4% SSF-BSG increased the digestibility of dry matter, starch, cellulose, glucans, and energy and did not affect protein digestibility. The present work showed the high potential of BSG to produce an added-value functional supplement with high carbohydrases activity and its potential contribution to the circular economy by improving the nutritional value of low-cost and sustainable ingredients that can be included in aquafeeds.

Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare.

PurposeTo evaluate complete blood count (CBC) parameters in the first week of life as predictive biomarkers for the development of retinopathy of prematurity (ROP).MethodsMulticenter, prospective, observational study of a cohort of preterm infants born with gestational age (GA) < 32 weeks or birth weight < 1500 g in eight Portuguese neonatal intensive care units. All demographic, clinical, and laboratory data from the first week of life were collected. Univariate logistic regression was used to assess risk factors for ROP and then multivariate regression was performed.ResultsA total of 455 infants were included in the study. The median GA was 29.6 weeks, and the median birth weight was 1295 g. One hundred and seventy-two infants (37.8%) developed ROP. Median values of erythrocytes (p < 0.001), hemoglobin (p < 0.001), hematocrit (p < 0.001), mean corpuscular hemoglobin concentration (p < 0.001), lymphocytes (p = 0.035), and platelets (p = 0.003) of the group of infants diagnosed with ROP any stage were lower than those without ROP. Mean corpuscular volume (MCV) (p = 0.044), red blood cell distribution width (RDW) (p < 0.001), erythroblasts (p < 0.001), neutrophils (p = 0.030), neutrophils-lymphocytes ratio (p = 0.028), and basophils (p = 0.003) were higher in the ROP group. Higher values of MCV, erythroblasts, and basophils remained significantly associated with ROP after multivariate regression.ConclusionIn our cohort, the increase in erythroblasts, MCV, and basophils in the first week of life was significantly and independently associated with the development of ROP. These CBC parameters may be early predictive biomarkers for ROP.

This study aimed to evaluate the effect of taurine (tau) supplementation to low fishmeal (FM) diets on growth performance, oxidative status, and immune response of European seabass juveniles. Four isoproteic (46% crude protein) and isolipidic (19% crude lipid) diets were formulated to contain either 25 or 12.5% FM and a mixture of plant feedstuffs, supplemented or not with 1% tau. Twelve groups of 20 fish (IBW = 9.4 g) were fed each diet for 9 weeks. Reduction of dietary FM from 25 to 12.5% impaired growth performance, feed efficiency, and protein efficiency ratio but had no effect on nitrogen retention (% N intake). Independently of FM level, dietary tau supplementation improved growth performance and nitrogen retention without affecting feed efficiency. Dietary FM level reduction increased liver G6PDH activity, but did not affect lipid peroxidation or activities of redox key enzymes. Contrarily, dietary tau supplementation decreased hepatic G6PDH and GPX activities and lipid peroxidation. Gene expression COX-2 was not affected either by FM or tau levels but TNF-α increased with the reduction of FM level but not with the tau level. Dietary tau supplementation decreased Casp3 and Casp9 expression regardless of dietary FM level. Overall, this study evidenced that dietary tau supplementation improved growth performance and antioxidant response and reduced intestine inflammatory and apoptosis processes.

Bacillus subtilis has been documented in the past years as an effective probiotic for different aquacultured species, with recognized beneficial effects on water quality, fish growth and immune status. Furthermore, its potential as a vaccine adjuvant has also been explored in different species. In the current work, we have used B. subtilis spores as delivery vehicles for the presentation of the VP2 protein from infectious pancreatic necrosis virus (IPNV). For this, the VP2 gene was amplified and translationally fused to the crust protein CotY. The successful expression of VP2 on the spores was confirmed by Western blot. We then compared the immunostimulatory potential of this VP2-expressing strain (CRS208) to that of the original B. subtilis strain (168) on rainbow trout ( Oncorhynchus mykiss ) leukocytes obtained from spleen, head kidney and the peritoneal cavity. Our results demonstrated that both strains significantly increased the percentage of IgM + B cells and the number of IgM-secreting cells in all leukocyte cultures. Both strains also induced the transcription of a wide range of immune genes in these cultures, with small differences between them. Importantly, specific anti-IPNV antibodies were detected in fish intraperitoneally or orally vaccinated with the CRS208 strain. Altogether, our results demonstrate B. subtilis spores expressing foreign viral proteins retain their immunomodulatory potential while inducing a significant antibody response, thus constituting a promising vaccination strategy.