A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms.

BACKGROUND: Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. OBJECTIVES: To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. METHODS: We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. RESULTS: The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. CONCLUSIONS: The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.

Bleaching efficacy and quality of life of different bleaching techniques - randomized controlled trial.

OBJECTIVES: To evaluate bleaching efficacy and oral health-related quality of life (ORHQoL) of three bleaching systems with similar hydrogen peroxide (HP) concentration for up to 6 months post-treatment. MATERIALS AND METHODS: A randomized controlled trial was designed with three parallel groups: group A - in-office 6% HP paint-on varnish; group B - at-home 6% HP with adaptable tray; group C - at-home 16% carbamide peroxide with custom tray. At three different stages (baseline, after bleaching, and 6-month follow-up), ORHQoL was evaluated by the OHIP-14 questionnaire and tooth color of the upper canines and central incisors were measured by two shade guides and a spectrophotometer (measuring CIE L*a*b* with respective color/whiteness differences - ΔE00/ΔWID). Results were presented as mean and 95% confidence intervals and statistical tests were performed appropriately, considering a significance level of α = 0.05. RESULTS: All groups presented significant color differences (P < 0.05) between all stages, with ΔE00/ΔWID surpassing the perceptibility threshold in 98% cases, with group C's results being significantly (P < 0.05) higher when compared to other groups, although with significantly (P < 0.05) higher values of color relapse. Significative ORHQoL improvements (P < 0.05) were detected after bleaching in a global analysis with no differences between techniques. CONCLUSIONS: All techniques presented bleaching efficacy, color stability, and improvements in ORHQoL up to 6 months post-treatment. CLINICAL SIGNIFICANCE: Clinicians may consider both at-home and in-office bleaching techniques with 6% HP to attain long-lasting satisfactory clinical results while producing positive changes in ORHQoL.

Mechanisms of bacterial membrane permeabilization by crotalicidin (Ctn) and its fragment Ctn(15-34), antimicrobial peptides from rattlesnake venom.

Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.

The mechanism of action of pepR, a viral-derived peptide, against Staphylococcus aureus biofilms.

OBJECTIVES: To investigate the mechanism of action at the molecular level of pepR, a multifunctional peptide derived from the Dengue virus capsid protein, against Staphylococcus aureus biofilms. METHODS: Biofilm mass, metabolic activity and viability were quantified using conventional microbiology techniques, while fluorescence imaging methods, including a real-time calcein release assay, were employed to investigate the kinetics of pepR activity at different biofilm depths. RESULTS: Using flow cytometry-based assays, we showed that pepR is able to prevent staphylococcal biofilm formation due to a fast killing of planktonic bacteria, which in turn resulted from a peptide-induced increase in the permeability of the bacterial membranes. The activity of pepR against pre-formed biofilms was evaluated through the application of a quantitative live/dead confocal laser scanning microscopy (CLSM) assay. The results show that the bactericidal activity of pepR on pre-formed biofilms is dose and depth dependent. A CLSM-based assay of calcein release from biofilm-embedded bacteria was further developed to indirectly assess the diffusion and membrane permeabilization properties of pepR throughout the biofilm. A slower diffusion and delayed activity of the peptide at deeper layers of the biofilm were quantified. CONCLUSIONS: Overall, our results show that the activity of pepR on pre-formed biofilms is controlled by its diffusion along the biofilm layers, an effect that can be counteracted by an additional administration of peptide. Our study sheds new light on the antibiofilm mechanism of action of antimicrobial peptides, particularly the importance of their diffusion properties through the biofilm matrix on their activity.

Synthesis and Characterization of Peptide-Chitosan Conjugates (PepChis) with Lipid Bilayer Affinity and Antibacterial Activity.

Antimicrobial peptides appear among innovative biopolymers with potential therapeutic interest. Nevertheless, issues concerning efficiency, production costs, and toxicity persist. Herein, we show that conjugation of peptides with chitosans can represent an alternative in the search for these needs. To increase solubility, deacetylated and degraded chitosans were prepared. Then, they were functionalized via N-succinimidyl- S-acetylthiopropionate or via glutathione (GSH), an endogenous peptide linker. To the best of our knowledge, it is the first time that GSH is used as a thiolating agent for the conjugation of peptides. Next, thiolated chitosans were conjugated through a disulfide bond with designed short-chain peptides, one of them derived from the antimicrobial peptide Jelleine-I. Conjugates and respective reaction intermediates were characterized by absorciometry, attenuated total reflectance-Fourier transform infrared, and 1H NMR. Zeta potential measurements showed the cationic nature of these biomacromolecules and their preferential partitioning to Gram-positive bacterial-like model membranes. In vitro investigation using representative Gram-positive and -negative bacteria ( Staphylococcus aureus and Escherichia coli, respectively) showed that the conjugation strategies lead to enhanced activity in relation to the unconjugated peptide and to the unconjugated chitosan. The obtained products showed selectivity toward S. aureus at low cytotoxicity as determined in NIH/3T3 cells. Overall, our study demonstrates that an appropriate choice of antimicrobial peptide and chitosan characteristics leads to increased antimicrobial activity of the conjugated product and represents a strategy to modulate the activity and selectivity of antimicrobials resorting to low-cost chemicals. The present proposal starts from less expensive raw materials (chitosan and short-chain peptide), is based on aqueous solvents, and minimizes the use of reactants with a higher environmental impact. The final biopolymer contains the backbone of chitosan, just 3-6% peptide derived from royal jelly and GSH, all of them considered safe for human use or as a physiological molecule.

Structural determinants conferring unusual long life in human serum to rattlesnake-derived antimicrobial peptide Ctn[15-34].

Ctn[15-34], a downsized version of the snake venom cathelicidin-like peptide crotalicidin (Ctn), shows an unusually high lifespan (t1/2 , approximately 12 h) in human serum, which significantly adds to its promise as an antimicrobial and antitumor agent. Herein we investigate the role of Ctn[15-34] structure on serum survival. Using a set of analogs, we show that C-terminal amidation, as well as the specific layout of the Ctn[15-34] sequence-a helical N-terminal domain followed by a hydrophobic domain-is crucial for slow degradation, and any change in their arrangement results in significantly lower t1/2 . Aside from the privileged primary structure, features such as self-aggregation can be ruled out as causes for the long serum life. Instead, studies in other protease-rich fluids suggest a key role for certain human serum components. Finally, we demonstrate that Ctn[15-34] is able to induce bacterial death even after 12-hour pre-incubation in serum, in agreement with the proteolytic data. Altogether, the results shed light on the uncommon stability of Ctn[15-34] in human serum and confirm its potential as an anti-infective lead.

Characterization of Tachyplesin Peptides and Their Cyclized Analogues to Improve Antimicrobial and Anticancer Properties.

Tachyplesin I, II and III are host defense peptides from horseshoe crab species with antimicrobial and anticancer activities. They have an amphipathic ß-hairpin structure, are highly positively-charged and differ by only one or two amino acid residues. In this study, we compared the structure and activity of the three tachyplesin peptides alongside their backbone cyclized analogues. We assessed the peptide structures using nuclear magnetic resonance (NMR) spectroscopy, then compared the activity against bacteria (both in the planktonic and biofilm forms) and a panel of cancerous cells. The importance of peptide-lipid interactions was examined using surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies showed that tachyplesin peptides and their cyclic analogues were most potent against Gram-negative bacteria and melanoma cell lines, and showed a preference for binding to negatively-charged lipid membranes. Backbone cyclization did not improve potency, but improved peptide stability in human serum and reduced toxicity toward human red blood cells. Peptide-lipid binding affinity, orientation within the membrane, and ability to disrupt lipid bilayers differed between the cyclized peptide and the parent counterpart. We show that tachyplesin peptides and cyclized analogues have similarly potent antimicrobial and anticancer properties, but that backbone cyclization improves their stability and therapeutic potential.

Mining viral proteins for antimicrobial and cell-penetrating drug delivery peptides.

MOTIVATION: The need for more effective and safer pharmaceuticals is a persistent quest. Microbial adaptations create the need to permanently develop new antimicrobials (AMPs), for instance. Similarly, intracellular delivery of drugs is still a challenge and translocation of membranes for drug delivery is an area of intense research. Peptides can be used both as AMP drug leads and drug carrier systems for intracellular delivery. Multifunctional proteins are abundant in viruses but, surprisingly, have never been thoroughly screened for bioactive peptide sequences. RESULTS: Using the AMPA and CellPPD online tools, we have evaluated the propensity of viral proteins to comprise AMP or cell-penetrating peptides (CPPs). Capsid proteins from both enveloped and non-enveloped viruses, and membrane and envelope proteins from enveloped viruses, in a total of 272 proteins from 133 viruses, were screened to detect the presence of potential AMP and CPP sequences. A pool of 2444 and 426 CPP and AMP sequences, respectively, were discovered. The capsids of flaviviruses are the best sources of these peptides reaching more than 80% of CPP sequence coverage per protein. Selected sequences were tested experimentally and validated the results. Overall, this study reveals that viruses form a natural multivalent biotechnological platform still underexplored in drug discovery and the heterogeneous abundance of CPP/AMP sequences among viral families opens new avenues in viral biology research.

Assessment of the Thermal Properties of Gypsum Plaster with Plastic Waste Aggregates.

Building material manufacturers must support new production models that encourage the manufacture of more efficient and sustainable products. This includes thinking about savings in the use of raw materials, a contribution to the energy efficiency of buildings during their useful life, and a reduction in the generation and deposit of waste in landfills. In this research, an analysis of the thermal properties of gypsum composites added with plastic waste is carried out using the most common methods, the steady state method and the transient plane source method, and the effect of water saturation on these composites is tested. The results show an improvement in the thermal performance of the composites (values reduced with respect to the reference by 4-7%), despite their heterogeneity, as well as a variation in the measurements carried out, depending on the method used for the measurements (variation up to 10%). It is also found that the degree of humidity negatively affects the thermal conductivity coefficient but, on the contrary, this coefficient is not altered in the composites with plastic waste, due to their lower hygroscopicity. Therefore, it is considered that the proposed eco-plasters are a good alternative to traditional plasters, with which to contribute to the achievement of the objectives of the current European directives on waste and circular economy.

Different Methods for Assessing Tooth Colour-In Vitro Study.

Colour assessment using digital methods can yield varying results, and it is important for clinicians to recognize the potential variability intra and inter-device. This study aimed to compare the L*a*b* values of VITA Classical (VC) and VITA Toothguide 3D-MASTER (VM) guides using two methods, SpectroShade (SS) and eLAB. Thirty-four measurements per tab were performed by a single operator across three batches of each guide. Intraclass correlation coefficients (ICC) between batches were calculated. Values <0.5, 0.5-0.75, 0.75-0.9, and >0.90 were classified as poor, moderate, good, and excellent reliability, respectively. Results were reported as mean and standard deviation of the L*a*b* values and respective colour differences (ΔE00) for each tab and method. Statistical analyses were performed with an independent t-test, α = 0.05. ICC values between batches were excellent for all L*a*b*, except for a* component in eLAB. There were statistically significant differences between methods in most L*a*b* values. The intra-device mean ΔE00 was 0.5 ± 0.6 for VC, 0.5 ± 0.8 for VM in SS, 1.1 ± 0.8 for VC, 1.1 ± 0.9 for VM in eLAB. The mean ΔE00 inter-device was 4.9 ± 1.7 for VC, 5.0 ± 1.7 for VM. Both methods demonstrated good internal consistency, with high ICC values and low intra-device colour differences, but exhibited high variability between methods, higher for a* the component.

Hydrogen Peroxide Diffusion through Dental Tissues-In Vitro Study.

Whitening products commonly utilize hydrogen peroxide (HP) as an active principle, which can penetrate dental tissues with potential side effects due to its low molecular weight. This study aimed to evaluate the HP diffusion of two in-office whitening products, namely 6% VivaStyle Paint On Plus (VS) and Opalescence Boost 40% (OP), in different tooth types. Additionally, the influence of the area of exposure, dental tissue thickness and pulp chamber volume was assessed. Each group consisted of eighteen intact anterior (A), premolar (PM) and molar (M) human teeth, and a positive pulpal pressure model was employed. The samples were analyzed using spectrophotometry, and results were expressed as the mean and 95% confidence interval. Statistical tests and linear regression models were appropriately applied at α = 5%. The total HP (µg) retrieved was as follows: VS-A, 1.333 [1.214, 1.452]; OP-A, 1.538 [1.457, 1.620]; VS-PM, 1.208 [1.123, 1.291]; OP-PM, 3.628 [3.401, 3.855]; VS-M, 2.560 [2.297, 2.823]; and OP-M, 4.197 [3.997, 4.396], with statistically significant differences in diffusion kinetics between whitening products for PM and M. Several HP concentrations attained a minimum cytotoxicity value of 2.22 µg/mL. The regression model shows that OP exposed the pulp chamber to 1.421 µg of HP more than that of VS. Different whitening products can cause cytotoxic HP concentrations in the pulp chamber, with a higher risk observed in molars.

Application of Ultrafiltration to Produce Sheep's and Goat's Whey-Based Synbiotic Kefir Products.

Membrane filtration technologies are the best available tools to manage dairy byproducts such as cheese whey, allowing for the selective concentration of its specific components, namely proteins. Their acceptable costs and ease of operation make them suitable for application by small/medium-scale dairy plants. The aim of this work is the development of new synbiotic kefir products based on sheep and goat liquid whey concentrates (LWC) obtained by ultrafiltration. Four formulations for each LWC based on a commercial kefir starter or traditional kefir, without or with the addition of a probiotic culture, were produced. The physicochemical, microbiological, and sensory properties of the samples were determined. Membrane process parameters indicated that ultrafiltration can be applied for obtaining LWCs in small/medium scale dairy plants with high protein concentration (16.4% for sheep and 7.8% for goats). Sheep kefirs showed a solid-like texture while goat kefirs were liquid. All samples presented counts of lactic acid bacteria higher than log 7 CFU/mL, indicating the good adaptation of microorganisms to the matrixes. Further work must be undertaken in order to improve the acceptability of the products. It could be concluded that small/medium-scale dairy plants can use ultrafiltration equipment to valorize sheep's and goat's cheese whey-producing synbiotic kefirs.

Cie L*A*B* Values in VITA Classical and VITA 3D Master by Two Dental Spectrophotometers.

PURPOSE: To compare the CIE L*a*b* values of two different dental shade guides using two shade-matching spectrophotometers. MATERIALS AND METHODS: SpectroShade Micro (SS) and Easyshade (ES) (VITA Zahnfabrik) were tested. One calibrated operator performed 30 measurements of each tab from three batches of the two guides, VITA Classical (VC) and VITA 3D Shade Master (VM). Receiver operating characteristic (ROC) curves and intraclass coefficients (ICC) between the different batches were calculated. Results were given as mean and SD of the L*a*b* values and the respective color differences according to the CIEDE 2000 formula (ΔE00) for each of the shade tabs in the two spectrophotometers and then analyzed with independent student t test (α = .05). RESULTS: A total of 1,440 and 2,610 measurements for VC and VM, respectively, were performed per spectrophotometer. There were statistically significant differences between the two devices for all L*a*b* values, with the exception of: L* for 3R1.5; a* for 2R1.5, 3L1.5, 2L2.5, 3M2, 3L2.5, 4L1.5, and 4L2.5; and b* for D3, 1M2, and 3M2. When assessing the same shade guide, differences in ΔE00 were detected between devices above the acceptability threshold (AT) (ΔE00 ≥ 1.8) for all shade tabs except for VC (C1, C2, D3, A3.5, C3, and A4) and VM (4M1, 3R2.5, 4L2.5, and 4M3). The overall mean of the interdevice ΔE00 was 2.2 ± 1.0 for VC and 2.5 ± 1.0 for VM. CONCLUSIONS: The two dental spectrophotometers presented high ICC and ROC values, which validates their indication as auxiliary tools. However, there are discrepancies in the hues D (VC) and R (VM) with interdevice variability when evaluated for the L*a*b* component. Int J Prosthodont 2023;36:e38-e52.

Quantitative Imaging of the Action of vCPP2319, an Antimicrobial Peptide from a Viral Scaffold, against Staphylococcus aureus Biofilms of a Clinical Isolate.

The formation of biofilms is a common virulence factor that makes bacterial infections difficult to treat and a major human health problem. Biofilms are bacterial communities embedded in a self-produced matrix of extracellular polymeric substances (EPS). In this work, we show that vCPP2319, a polycationic peptide derived from the capsid protein of Torque teno douroucouli virus, is active against preformed Staphylococcus aureus biofilms produced by both a reference strain and a clinical strain isolated from a diabetic foot infection, mainly by the killing of biofilm-embedded bacteria. The direct effect of vCPP2319 on bacterial cells was imaged using atomic force and confocal laser scanning microscopy, showing that the peptide induces morphological changes in bacterial cells and membrane disruption. Importantly, vCPP2319 exhibits low toxicity toward human cells and high stability in human serum. Since vCPP2319 has a limited effect on the biofilm EPS matrix itself, we explored a combined effect with α-amylase (EC 3.2.1.1), an EPS matrix-degrading enzyme. In fact, α-amylase decreases the density of S. aureus biofilms by 2.5-fold. Nonetheless, quantitative analysis of bioimaging data shows that vCPP2319 partially restores biofilm compactness after digestion of the polysaccharides, probably due to electrostatic cross-bridging of the matrix nucleic acids, which explains why α-amylase fails to improve the antibacterial action of the peptide.

Sheep's Butter and Correspondent Buttermilk Produced with Sweet Cream and Cream Fermented by Aromatic Starter, Kefir and Probiotic Culture.

Small ruminant dairy products are common in some Mediterranean countries, in the Middle East and Africa, and can play a particular role in the development of rural areas. Butter has been the object of few research studies aimed at evaluating its potential as a vehicle for probiotic microorganisms. Moreover, the recovery of fermented buttermilk with functional properties can be considered an excellent opportunity to value this dairy byproduct. Therefore, the purpose of the present work was to develop different sheep butters and respective buttermilks after cream fermentation by: (1) a mesophilic aromatic starter (A); (2) a kefir culture (K); and (3) a mixture of probiotic bacteria (P). The butters and buttermilk produced with fermented cream were compared with non-fermented sweet cream (S) butter or buttermilk, respectively, regarding their physicochemical, microbiological and sensory characteristics. The adjusted production (%, w/v) obtained for butter were: S (44.48%), A (36.82%), K (41.23%) and P (43.36%). S, A and K butters had higher solids, fat and ashes contents than P butter. The probiotic butter had a total fat of ca. 75% (w/w), below the legal limits, while all others had fat levels above 81.5%. In all samples, the pH decreased and the acidity increased over 90 days of refrigerated storage. These variations were more evident in the P butter, which agrees with the highest lactic acid bacteria counts found in this sample. Differences in color between samples and due to storage time were also observed. In general, the butter samples tended to become darker and yellower after the 60th day of storage. Texture analysis showed comparable results between samples and greater hardness was observed for the P butter, most probably due to its higher relative saturated fatty acids content (66.46% compared to 62−64% in S, A and K butters). Regarding rheological properties, all butters showed pseudoplastic behavior, but butter P had the lowest consistency index (249 kPa.sn−1). The probiotic butter and the corresponding buttermilk had viable cell counts greater than 7 Log CFU/g, indicating their suitability as probiotic carriers. All products were well accepted by consumers and small, but non-significant, differences (p > 0.05) were observed in relation to the sensory parameters evaluated. In general, it can be concluded that the use of adequate starter cultures can allow the production of innovative and potentially healthier products, alongside the valorization of dairy byproducts, improving the income of small-scale producers.

Effect of Extraction Method on the Bioactive Composition, Antimicrobial Activity and Phytotoxicity of Pomegranate By-Products.

Pomegranate by-products can be an asset to the food industry due to the richness in bioactive and antimicrobial compounds. This work studied the influence of conventional solvent and sonication-assisted extraction methods on the bioactive profile, antimicrobial properties, and phytotoxicity effect of the peels and seeds extracts from Acco, Big Full, and Wonderful pomegranate cultivars. The bioactive composition of the extracts was evaluated for the content of total phenolics, total flavonoids, and antioxidant activity (expressed as the half-maximal inhibitory concentration-IC50) by spectrophotometric methods, while the tannins were determined by titration and the anthocyanins were estimated by the pH-differential method. For the evaluation of the antimicrobial activity, the disk diffusion method of Kirby-Bauer was adapted through inhibition halos against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and Yarrowia lipolytica. The extracts' phytotoxicity was evaluated in vitro on garden-cress seeds. Extracts from conventional extraction were richer in total phenolics, expressed as gallic acid equivalents (0.16-0.73 mg GAE/mg extract), while those from sonication-assisted extraction had higher contents of total flavonoids, expressed as catechin equivalents (0.019-0.068 mg CATE/mg extract); anthocyanins, expressed as cyanidin-3-glucoside (0.06-0.60 µg C3G/mg, dry basis); and antioxidant activity (IC50, 0.01-0.20 mg/mL). All extracts were more effective against Gram-positive bacteria and yeasts than Gram-negative bacteria. In general, the sonication-assisted extracts led to higher inhibition halos (8.7 to 11.4 mm). All extracts presented phytotoxicity against garden-cress seeds in the tested concentrations. Only the lowest concentration (0.003 mg/mL) enabled the germination of seeds and root growth, and the sonication-assisted extracts showed the highest Munoo-Liisa vitality index (51.3%). Overall, sonication-assisted extraction obtained extracts with greater bioactive and antimicrobial potential and less phytotoxicity.

Microbial Characterization of Yellow Curing Process of Codfish.

Yellow cured codfish has a typical yellow colour, distinctive taste, and low salt content due to its special curing process of the raw salted codfish involving several soaks in water of the raw salted codfish, alternated with drying steps. The purpose of this study was to assess the main functional groups of bacteria involved in this process and relate them with physicochemical properties of the product. A total of 28 codfish from Iceland were supplied by two local companies. Seven stages of the curing process were analyzed. From each of these seven stages, four fish samples were collected to carry out the microbial and physicochemical analyses (moisture, salt content, pH, total volatile basic nitrogen (TVB-N), and trimethylamine nitrogen (TMA-N)). Bacteria counts were performed using the MPN method and adequate culture media for aerobic, proteolytic, sulphite-reducing, biogenic amine, and trimethylamine-producing and ammonifying bacteria. Strains isolated from the highest dilutions with microbial growth were used to characterize the predominant bacteria. The results showed that total aerobic counts increased from 3.9 log MPN/g in raw salted codfish to 5.9 log MPN/g in the final. Proteolytic, ammonifying, and trimethylamine bacteria producers also increased to 8, 7.5, and 6.5 log MPN/g, respectively. The salt content decreases (from 17% until 8%) and moisture increases (53% until 67%) during the salted-raw-codfish soaking, favoring sulphite-reducing and biogenic amine-producing species, confirming that desalting enhances potential spoilers. The subsequent drying step benefits proteolytic, ammonifying, and trimethylamine-producing bacteria, with a corresponding non-protein-nitrogen content (TVB-N and TMA-N) increase. The dominant bacteria during yellow curing belong to the genera Staphylococcus, Psychrobacter, Pseudomonas, and Alcaligenes with a clear positive correlation between the content of Staphylococcus and Psychrobacter and TVB-N and TMA-N concentration. Staphylococcus spp. are the dominant bacteria in the steps where the product has a higher salt concentration; thus, it could be particularly useful as an indicator to control the industrially yellow curing process and could have an important role in the development of the final characteristics of this product.

Hydrogen Peroxide Release Kinetics of Four Tooth Whitening Products-In Vitro Study.

Tooth whitening efficacy can be influenced by several factors, of which concentration and application time are two of the most important. This in vitro study aimed to evaluate the initial content and release kinetics of the hydrogen peroxide (HP) content, or the carbamide peroxide (CP) content as converted to its HP equivalent, of four tooth whitening products with different concentrations (6% HP, 16% CP, 10% CP, and 5% CP). Titrations with Cerium Sulphate IV were performed to determine HP concentration. HP release kinetics were evaluated by a spectrophotometric technique. The results were expressed as the mean values and 95% confidence interval of the percentage of hydrogen peroxide content during release kinetics. One sample t-test, one-way ANOVA, Tukey post hoc testing, and Pearson correlation testing were used, as appropriate, with a significance level of α = 0.05. The concentration of titrated HP was higher than that indicated by the manufacturers in all tested products (p < 0.01). At the minimum application times indicated by the manufacturers, all products released at least 85% of HP content; the gel containing 10% CP registered the lowest release at 85.49 (81.52-89.46). There was a significant HP release in all products during the application times indicated by the manufacturers. Further studies are needed to assess in vitro release kinetics.

Patients and Health Professional's Perspective of Functional Mobility in Parkinson's Disease.

Background: Functional mobility (FM) is the person's ability to move to accomplish daily living tasks and activities. FM limitations are common in Parkinson's disease, increase with disease progression, and can be highly disabling. Although several studies in Parkinson's disease (PD) field use this concept, only recently, a formal definition has been proposed. Objective: We aimed to explore patient's and health professional's perspectives of FM in PD. Methods: A focus group methodology has been used. Four focus groups, with a total of 10 patients and 10 health professionals, were performed. Six patients were early stage and four advanced stage. The health professional's group was composed of five neurologists and five physiotherapists. The suitability of the new concept, the impact of FM limitations in PD patient's daily routine, and the potential benefit of walking aids have been discussed. Results: All participants were able to provide a spontaneous definition of FM, matching with the proposed concept. All agreed that PD affects patient's FM, increasing the limitations with disease progression, and with the existence of a serious prejudice with walking aids that hinders its use. Early-stage patient's perspective seems to be more in line with neurologist's perspective, while the views of advanced-stage patients were closer to physiotherapist's views. Conclusion: FM concept was considered as intuitive and useful. FM limitations have an important physical and social impact in the advanced stage of the disease. Although patients and health professionals acknowledge walking aid's benefit improving patient's FM, the prejudice associated with this type of tools limits its recommendation and use.

Design of bioactive peptides derived from CART sequence isolated from the toadfish Thalassophryne nattereri.

The emergence of bacterial resistance due to the indiscriminate use of antibiotics warrants the need for developing new bioactive agents. In this context, antimicrobial peptides are highly useful for managing resistant microbial strains. In this study, we report the isolation and characterization of peptides obtained from the venom of the toadfish Thalassophryne nattereri. These peptides were active against Gram-positive and Gram-negative bacteria and fungi. The primary amino acid sequences showed similarity to Cocaine and Amphetamine Regulated Transcript peptides, and two peptide analogs-Tn CRT2 and Tn CRT3-were designed using the AMPA algorithm based on these sequences. The analogs were subjected to physicochemical analysis and antimicrobial screening and were biologically active at concentrations ranging from 2.1 to 13 µM. Zeta potential analysis showed that the peptide analogs increased the positive charge on the cell surface of Gram-positive and Gram-negative bacteria. The toxicity of Tn CRT2 and Tn CRT3 were analyzed in vitro using a hemolytic assay and tetrazolium salt reduction in fibroblasts and was found to be significant only at high concentrations (up to 40 µM). These results suggest that this methodological approach is appropriate to design novel antimicrobial peptides to fight bacterial infections and represents a new and promising discovery in fish venom.