Effect of by-products from the dairy industry as alternative inducers of recombinant ß-galactosidase expression.

OBJECTIVE: The aim of the present study was to evaluate the efficiency of lactose derived from cheese whey and cheese whey permeate as inducer of recombinant Kluyveromyces sp. ß-galactosidase enzyme produced in Escherichia coli. Two E. coli strains, BL21(DE3) and Rosetta (DE3), were used in order to produce the recombinant enzyme. Samples were evaluated for enzyme activity, total protein content, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis after induction with isopropyl-ß-D-1-thiogalactoside (IPTG) (0.05 and 1 mM) and lactose, cheese whey, and cheese whey permeate solutions (1, 10, and 20 g/L lactose) at shake-flask cultivation, and whey permeate solution (10 g/L lactose) at bioreactor scale. RESULTS: The highest specific activities obtained with IPTG as inducer (0.05 mM) after 9 h of induction, were 23 and 33 U/mgprotein with BL21(DE3) and Rosetta(DE3) strains, respectively. Inductions performed with lactose and cheese whey permeate (10 and 20 g/L lactose) showed the highest specific activities at the evaluated hours, exhibiting better results than those obtained with IPTG. Specific activity of recombinant ß-galactosidase using whey permeate (10 g/L lactose) showed values of approximately 46 U/mgprotein after 24-h induction at shake-flask study, and approximately 26 U/mgprotein after 16-h induction at bench bioreactor. CONCLUSIONS: The induction with cheese whey permeate was more efficient for recombinant ß-galactosidase expression than the other inducers tested, and thus, represents an alternative form to reduce costs in recombinant protein production.

Influence of apitoxin and melittin from Apis mellifera bee on Staphylococcus aureus strains.

The antibacterial activities of apitoxin, a venom produced by Apis mellifera bee, and melittin, an antimicrobial peptide from apitoxin, were tested against planktonic and biofilm states of Staphylococcus aureus methicillin-resistant (MRSA), including clinical, and enterotoxin-producing isolates. Also, the synergism of apitoxin and melittin in combination with oxacillin were evaluated as well. The induced morphological changes on S. aureus cells of both products were detected by transmission electronic microscopy (TEM). The minimum inhibitory concentration (MIC) values were 7.2 µg/mL, and 6.7 µg/mL, for apitoxin and melittin, respectively. The minimum bactericidal concentration (MBC) values were 28.7 µg/mL, and 26 µg/mL for apitoxin and melittin, respectively. The time-kill curve assays of apitoxin or melittin with oxacillin exhibited bactericidal synergism against MRSA isolates. TEM images showed cell distortion, cell disintegration with leakage of cytoplasmic content and loss of cytoplasm content. However, apitoxin and melittin did not interfere with staphylococcal enterotoxin production or release. Thus, apitoxin and melittin are potential agents against MRSA that can serve as possible models for new antibacterial drugs.

Annexin A1-derived peptide Ac2-26 in a pilocarpine-induced status epilepticus model: anti-inflammatory and neuroprotective effects.

BACKGROUND: The inflammatory process has been described as a crucial mechanism in the pathophysiology of temporal lobe epilepsy. The anti-inflammatory protein annexin A1 (ANXA1) represents an interesting target in the regulation of neuroinflammation through the inhibition of leukocyte transmigration and the release of proinflammatory mediators. In this study, the role of the ANXA1-derived peptide Ac2-26 in an experimental model of status epilepticus (SE) was evaluated. METHODS: Male Wistar rats were divided into Naive, Sham, SE and SE+Ac2-26 groups, and SE was induced by intrahippocampal injection of pilocarpine. In Sham animals, saline was applied into the hippocampus, and Naive rats were only handled. Three doses of Ac2-26 (1 mg/kg) were administered intraperitoneally (i.p.) after 2, 8 and 14 h of SE induction. Finally, 24 h after the experiment-onset, rats were euthanized for analyses of neuronal lesion and inflammation. RESULTS: Pilocarpine induced generalised SE in all animals, causing neuronal damage, and systemic treatment with Ac2-26 decreased neuronal degeneration and albumin levels in the hippocampus. Also, both SE groups showed an intense influx of microglia, which was corroborated by high levels of ionised calcium binding adaptor molecule 1(Iba-1) and monocyte chemoattractant protein-1 (MCP-1) in the hippocampus. Ac2-26 reduced the astrocyte marker (glial fibrillary acidic protein; GFAP) levels, as well as interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and growth-regulated alpha protein (GRO/KC). These effects of the peptide were associated with the modulation of the levels of formyl peptide receptor 2, a G-protein-coupled receptor that binds to Ac2-26, and the phosphorylated extracellular signal-regulated kinase (ERK) in the hippocampal neurons. CONCLUSIONS: The data suggest a neuroprotective effect of Ac2-26 in the epileptogenic processes through downregulation of inflammatory mediators and neuronal loss.

Antibacterial and anti-biofilm activities of cinnamaldehyde against S. epidermidis.

The search for new antimicrobial drugs has been necessary due to the increased bacterial resistance to antibiotics currently in use, and natural products play an important role in this field. The aim of this study was to evaluate the in vitro effect of cinnamaldehyde on S. epidermidis strains, biofilm set-up prevention, as well as its effect on pre-established biofilms. The minimum inhibitory concentration (MIC) ranged from 300 to 500 µg/mL, and the minimum bactericidal concentration (MBC) from 400 to 600 µg/mL. The biofilm inhibitory concentration and biofilm eradication concentration values were four-fold (clinical isolate) and eight-fold (ATCC strain) greater than the concentration required to inhibit planktonic growth. Sub-inhibitory concentrations of cinnamaldehyde attenuated biofilm formation of S. epidermidis strains on polystyrene microtiter plates. The combination of cinnamaldehyde and linezolid was able to inhibit S. epidermidis with a bactericidal effect. Further investigation of the mechanism of action of cinnamaldehyde revealed its effect on the cell membrane permeability, and confocal laser scanning microscopy (CLSM) images illustrated the impact of cinnamaldehyde in the detachment and killing of existing biofilms. Thereby, our data confirmed the ability of cinnamaldehyde to reduce bacterial planktonic growth of S. epidermidis, inhibiting biofilm formation and eradicating pre-formed biofilm.

Short communication: Inhibitory activities of the lantibiotic nisin combined with phenolic compounds against Staphylococcus aureus and Listeria monocytogenes in cow milk.

We aimed to investigate the antibacterial activities of carvacrol, thymol, eugenol, cinnamaldehyde, and lantibiotic nisin against standard bacterial strains of the milk pathogens Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 15313 in cow milk. The minimum inhibitory concentrations (MIC) of these substances were recorded. The synergistic effects were also assessed in culture medium (time kill curve) and in a food model (cow milk) during the storage period (4 °C for 6 d) after inoculation with S. aureus and L. monocytogenes individually by combining nisin and the phenolic compounds at proportions of 1/4 + 1/4 the MIC (determined in a previous in vitro assay) in the culture medium and 1/4 + 1/4 of MIC in the food model. Inhibitory activities of nisin and the tested compounds, as well as synergism in the combinations, were found against both bacteria assayed. Bacteriostatic effects were found with all combinations and a significant difference in L. monocytogenes reduction was found compared with the control assays. Thus, the antibacterial activity of nisin combined with phenolic compounds was confirmed against these pathogenic bacteria that are important in the milk industry, or more broadly in food science, with potential applications for milk preservation.

S-nitrosothiols as nitrite alternatives: Effects on residual nitrite, lipid oxidation, volatile profile, and cured color of restructured cooked ham.

This study evaluated the use of the S-nitrosothiols, S-nitroso-N-acetylcysteine (NAC-SNO) and S-nitroso-N-acetylcysteine ethyl ester (NACET-SNO), at different concentrations (25-300 mg nitrite equivalent - NEq/kg) as sodium nitrite substitutes in restructured cooked hams. The pH value and instrumental cured color were not affected by the type or amount of curing agent used. Products with 25 and 50 mg/kg ingoing nitrite had lower thiobarbituric acid-reactive substance values than those with equimolar amounts of S-nitrosothiols. Products with >150 mg NEq/kg of S-nitrosothiols had residual nitrite similar to 50 mg/kg nitrite, and this resulted in the same volatile compound profile as nitrite added in equimolar amounts. A 300 mg NEq/kg of S-nitrosothiols was required to obtain a similar and minimally stable cured pink color perception as sliced samples with 50-150 mg/kg added nitrite. The results obtained reinforce the great potential of both alternative curing agents in the complete replacement of nitrite by equimolar amounts in restructured cooked products; however, differences in cured color stability should be considered.

Technological and sensory characteristics in development of innovative symbiotic boneless dry-cured lamb meat snack.

Novel shelf-stable and high-protein meat products that are affordable, convenient, and healthy are hot topic in current food innovation trends. To offer technological databases for developing new functional lamb meat products, this study aimed to evaluate the technological and sensory aspects of dry-cured lamb meat snacks incorporated with the probiotic culture Lactobacillus paracasei and the prebiotic lactulose. Four formulations were analyzed: control (without prebiotic or probiotic); PREB (with 2% lactulose); PROB (with 107 CFU/g of L. paracasei); and SYMB (with 2% lactulose and 107 CFU/g of L. paracasei). Fitted curves revealed that weight-loss behavior during snack ripening was not affected (P > 0.05) by treatments. Snack moisture, water activity, pH, titratable acidity, lipid oxidation, and residual nitrite were affected (P < 0.05) only by ripening time. The target probiotic strain stood out against competitive flora and was detected at 107 CFU/g in the snack-supplemented formulations (PROB and SYMB). In snacks supplemented with prebiotics (PREB and SYMB), the lactulose content was maintained at 2.17%. Significant differences were not observed in the chemical composition, texture profiles, and CIE color indices between the proposed functional snacks and the control. In addition to texture, flavor, and overall impression evaluation, only color attributes were positively impacted (P < 0.05) in the acceptance and multiple comparison tests against the control. The proposed formulation and bench process parameters produced potential nutritionally and sensory-appreciated, microbiologically stable, and safe (multi-hurdle perspective) functional high-protein restructured lamb snacks.

TNF-α blockage prevents late neurological consequences of Zika virus infection in mice.

Zika virus (ZIKV) is a neurotropic Orthoflavivirus that causes a myriad of neurological manifestations in newborns exposed in uterus. Despite the devastating consequences of ZIKV on the developing brain, strategies to prevent or treat the consequences of viral infection are not yet available. We previously showed that short-term treatment with the TNF-α neutralizing monoclonal antibody. Infliximab could prevent seizures at acute and chronic stages of ZIKV infection, but had no impact on long-term cognitive and motor dysfunction. Due to the central role of inflammation in ZIKV-neuropathology, we hypothesized that prolonged treatment with the anti-TNF-α monoclonal antibody Infliximab could provide complete rescue of long-term behavioral deficits associated with neonatal ZIKV infection in mice. Here, neonatal (post-natal day 3) Swiss mice were submitted to subcutaneous (s.c.) injection of 106 PFU of ZIKV or mock medium and were then treated with Infliximab (20 µg/day) or sterile saline intraperitoneally (i.p.), for 40 days starting on the day of infection, and behavioral assessment started at 60 days post-infection (dpi). Infliximab prevented ZIKV-induced cognitive and motor impairments in mice. In addition, microgliosis and cell death found in mice following ZIKV infection were partially reversed by TNF-α blockage. Altogether, these results suggest that TNF-α-mediated inflammation is central for late ZIKV-induced behavioral deficits and cell death and strategies targeting this cytokine may be promising approaches to treat subjects exposed to the virus during development.

Predictive factors for the diagnosis of permanent congenital hypothyroidism and its temporal changes in Sergipe, Brazil - A real-life retrospective study.

Objective: Congenital hypothyroidism (CH) can be permanent (PCH) or transient (TCH). While the importance of thyroxine in myelination of the brain is undisputed, the benefits to neurodevelopmental outcomes of TCH treatment are controversial. Our objectives were to determine predictive factors for PCH and verify its prevalence changes over time. Subjects and methods: A total of 165 children were evaluated at 3 years of age to verify the diagnosis of PCH. 130 were submitted to a two-step cluster analysis, with the aim of grouping them into homogeneous clusters. The mean incidence of PCH and TCH was calculated from 2004 to 2010 and 2011 to 2015. Results: Sixty-six children were diagnosed with PCH, and 99 were diagnosed with TCH. Eighty-one percent of PCH children and all TCH children with thyroid imaging had glands in situ. Eighty children (61.5%) were in Cluster 1, 8 children (6.2%) were in Cluster 2 and 42 children (32.3%) were in Cluster 3. No children had PCH in Cluster 1, while 87.5% of children in Cluster 2 and all children in Cluster 3 had PCH. The most important predictor for PCH was the initial serum TSH, which was marginally higher in importance than the blood spot TSH, followed by the initial serum free T4. The mean incidence of PCH (odds ratio: 1.95, 95% CI 1.36 to 2.95, p < 0.0001) and TCH (odds ratio 1.33, 95%, CI 1.02 to 1.77, p = 0,038) increased over time. Conclusion: The most important PCH predictors are the initial serum TSH and the blood spot TSH. The mean incidence of both PCH and TCH in our series increased.

Dental arches in inherited severe isolated growth hormone deficiency.

OBJECTIVES: The growth of the dental arches depends on GH and insulin-like growth factor type 1 (IGF1), but the consequences of GH deficiency (GHD) on their growth are still unclear, probably due to the acquired etiology of GHD in most described series, often associated with additional pituitary deficits (thyrotrophic, corticotrophic and gonadotrophic hormones), and imperfections of related replacement therapies, which may affect the dental arch growth. To avoid these limitations, we took advantage of a unique cohort of subjects with isolated GH deficiency (IGHD) due the same mutation in the GH releasing hormone receptor gene, living with very low serum GH and low to undetectable circulating IGF1 levels. Our purpose was to analyze the dimensions of maxillary and mandibular dental arches. METHODS: 22 adult IGHD (15 untreated and 7 previously partially treated with GH) and 33 controls were enrolled in a cross-sectional study using the Ortho Insight 3D and MeshMixer software, RESULTS: In untreated IGHD subjects all maxillary arch measures were smaller than controls, while among mandibular arches, only the mandibular canine width and the mandibular arch length were reduced. In partially GH treated subjects only the palate depth, the maxillary canine width, the maxillary and mandibular arch lengths remained smaller than controls. CONCLUSIONS: IGHD reduces the growth of maxillary arch to a greater degree than the mandibular arch, suggesting different control of superior and inferior dental arches. GH treatment increases some of these measures.

Growth of teeth and bones in adult subjects with congenital untreated isolated growth hormone deficiency.

OBJECTIVE: To understand the growth of teeth and mandibular and maxillary bones in subjects with isolated growth hormone deficiency (IGHD). MATERIAL AND METHODS: Mesiodistal tooth width of 28 maxillary and mandibular dental models of 14 adult IGHD subjects (9 men) were digitalized and compared to 40 models of 20 normal-statured controls (11 men). The mean SDS of the maxillary and mandibular teeth were compared with height, cephalic perimeter, total anterior facial height, total maxillary and mandibular length, and maxillary and mandibular arches. RESULTS: All average mesiodistal dimensions in absolute values of the 14 dental pairs were reduced in the IGHD group. Eight of 28 (28.6%) mesiodistal dimensions in IGHD subjects of both sexes had an average SDS below -2, thirteen of them (46.4%) had mean SDS between -1 and - 2, and seven of them (25.0%) had SDS above -1. The highest SDS values were the upper lateral incisor (-0.32 in women), and the upper canine (-0.91 in men). The lowest SDS values were the 2nd upper molar (-3.51 in men), and the 2nd upper premolar (-2.64 in women). The ascending order of the mean SDS was height, total maxillary length, total mandibular length, total anterior height of the face, cephalic perimeter, the maxillary arches width, the mesiodistal width of the mandibular teeth, the mesiodistal width of the maxillary teeth and the mandibular arches width. CONCLUSIONS: Reduction in mesiodistal width is present in untreated IGHD adults with magnitude of tooth size reduction being lower than height, cephalic perimeter, total anterior facial height, and most jaw measurements. IGHD abolishes the sexual dimorphism in mesiodistal dental measures.

Art and science: impact of semioccluded vocal tract exercises and choral singing on quality of life in subjects with congenital GH deficiency.

Objective: Currently, not much is known about the interactions between voice and growth hormone (GH). We have described large kindred with isolated GH deficiency (IGHD) due to a GHRH receptor mutation, resulting in severe short stature and high-pitched voice. These IGHD individuals have little interest in GH treatment, as they consider themselves "short long-lived people", rather than patients. Interestingly, they report normal general quality of life, but they rate their Voice-Related Quality of Life (V-RQOL) as low. Here, we assessed the social and auditory-perceptual impacts of artistic-intervention voice therapy with semioccluded vocal tract exercises (SOVTE) and choral singing, on their voices. Methods: Seventeen GH-naïve adult IGHD individuals were enrolled in a single-arm interventional pre-post study with 13 weekly sessions of choir singing over 90 days. Outcome measures were V-RQOL scores, self-assessment of voice, and auditory-perceptual analysis (GRBAS scale, G: grade of the severity of dysphonia; R: roughness; B: breathiness; A: asthenia; and S: strain). Results: Marked improvements in total (p = 0.0001), physical (p = 0.0002), and socioemotional (p = 0.0001) V-RQOL scores and in self-assessment of voice (p = 0.004) were found. The general grades of vocal deviation (p = 0.0001), roughness (p = 0.0001), breathiness (p = 0.0001) and strain (p = 0.0001) exhibited accentuated reductions. Conclusion: Voice therapy with semioccluded vocal tract exercises and choral training improved social impact and perceptual voice assessments in IGHD subjects and markedly improved their voice-related quality of life. This is particularly important in a setting where GH replacement therapy is not widely accepted.

Production of beta-galactosidase fused to a cellulose-binding domain for application in sustainable industrial processes.

This study aimed to produce and characterize a recombinant Kluyveromyces sp. ß-galactosidase fused to a cellulose-binding domain (CBD) for industrial application. In expression assays, the highest enzymatic activities occurred after 48 h induction on Escherichia coli C41(DE3) strain at 20 °C in Terrific Broth (TB) culture medium, using isopropyl ß-d-1-thiogalactopyranoside (IPTG) 0.5 mM (108.77 U/mL) or lactose 5 g/L (93.10 U/mL) as inducers. Cultures at bioreactor scale indicated that higher product yield values in relation to biomass (2000 U/g) and productivity (0.72 U/mL.h) were obtained in culture media containing higher protein concentration. The recombinant enzyme showed high binding affinity to nanocellulose, reaching both immobilization yield and efficiency values of approximately 70% at pH 7.0 after 10 min reaction. The results of the present study pointed out a strategy for recombinant ß-galactosidase-CBD production and immobilization, aiming toward the application in sustainable industrial processes using low-cost inputs.

Formant Frequencies, Cephalometric Measures, and Pharyngeal Airway Width in Adults With Congenital, Isolated, and Untreated Growth Hormone Deficiency.

OBJECTIVE: Adult subjects with isolated growth hormone deficiency (IGHD) due to a mutation in the growth hormone releasing hormone receptor gene exhibit higher values formant frequencies. In normal subjects, a significant negative association between the formant frequencies and the reduction of linear craniofacial measurements, especially of maxilla and mandible, has been reported. This suggests smaller pharyngeal width, despite low prevalence of obstructive sleep apnea syndrome. Here we evaluate their pharyngeal airway width, its correlation with vowel formant frequencies, and the correlation between them and the craniofacial measures. SUBJECTS AND METHODS: A two-step protocol was performed. In the first case-control experiment, aimed to assess the pharyngeal width, we compared nine adult IGHD and 36 normal statured controls. Both upper and lower pharyngeal widths were measured. The second step (assessment of pharyngeal width) was performed only in the IGHD group. RESULTS: Upper and lower pharyngeal widths were similar in IGHD and controls. In IGHD subjects, the lower pharyngeal width exhibited a negative correlation with F1 [a] and a positive correlation with mandibular length. There were negative correlations between F1 and F2 with linear and positive correlations with the angular measures. CONCLUSIONS: Pharyngeal airway width is not reduced in adults with congenital, untreated lifetime IGHD, contributing to the low prevalence of obstructive sleep apnea syndrome. The formant frequencies relate more with cephalometric measurements than with the pharyngeal airway width. These findings exemplify the consequences of lifetime IGHD on osseous and nonosseous growth.

Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using ß-galactosidase as a model.

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using ß-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant ß-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized ß-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant ß-galactosidase, showing its potential application in other high-molecular-weight enzymes.

Endodontic approach in a replanted tooth with an immature root apex and chronic apical periodontitis: a case report.

This study describes the clinical steps taken in the treatment of a patient who had an avulsed right upper central incisor that presented with incomplete root development and chronic apical periodontitis. A 7-year-old boy was referred from a private dentist to a dental office specializing in endodontics. The tooth had remained in a dry environment for 20 minutes, and tooth replantation was performed at an emergency appointment. After clinical and radiographic examinations, root canal decontamination was performed, followed by several changes in intracanal calcium hydroxide medication. Blood clot formation was attempted, but bleeding within the root canal was insufficient; therefore, we opted for an intracanal medication change to stimulate mineralized tissue formation in the apical region. Root obturation was performed 45 days after the last change of intracanal medication, and clinical, radiographic, and tomographic follow-up examinations were performed at 3, 6, 18, and 40 months after the endodontic intervention. The increase in thickness and length of the root structure and the absence of root resorption were verified through follow-up examinations. Therefore, it was concluded that the procedures used were successful for tooth replantation.

Microbial ß-Galactosidases of industrial importance: Computational studies on the effects of point mutations on the lactose hydrolysis reaction.

Hydrolysis efficiency of ß-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial ß-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnßGal), Aspergillus oryzae (AoßGal), Bacillus circulans (BcßGal), Bifidobacterium bifidum (BbßGal), and Kluyveromyces lactis (KlßGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnßGal), Asn140Ala and Asn199Ala (AoßGal), Arg111Ala and Glu355Ala (BcßGal), Arg122Ala and Phe358Ala (BbßGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlßGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcßGal, BbßGal, and KlßGal. The double mutations in BcßGal and BbßGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlßGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of ß-galactosidases and rendering them promising for industrial applications.

Comparative Proteomics of Methicillin-Resistant Staphylococcus aureus Subjected to Synergistic Effects of the Lantibiotic Nisin and Oxacillin.

We investigated the responses and mechanisms of action of methicillin-resistant Staphylococcus aureus (MRSA) metabolism when exposed under sublethal concentrations of the synergistic antibacterial combination of nisin + oxacillin (» of maximum sublethal concentration) and sublethal concentrations of oxacillin only and nisin only. A total of 135 proteins were identified, showing an alteration in the expression of 85 proteins when treatment was compared with untreated bacteria (control). When the bacteria were treated using the combination, there was an increase in the expression of proteins related to resistance (e.g., beta-lactamase) and also in the ones involved in protein synthesis, and there was a decrease in the expression of proteins related to stress and alterations in proteins related to bacterial energy metabolism. Bacterial oxidative stress showed that the combination was able to induce oxidative stress (p < 0.05) and increase enzyme activities and lipid hydroperoxide levels compared with individual treatments. The analysis of cell ultrastructure showed damage in MRSA, especially on the bacterial wall and the plasma membrane, with cell lysis and death. Thus, the changes caused by these treatments affected different proteins related to the bacterial biological processes and signaling pathways such as cell division, structure, stress, regulation, bacterial resistance, protein synthesis, gene expression, energetic metabolism, and virulence. It was observed that synergism among antimicrobials has high potential in therapeutic use and may reduce the required amounts of antibacterial substances in addition to being effective on different targets in bacterial cells.

Proteomic analysis and antibacterial resistance mechanisms of Salmonella Enteritidis submitted to the inhibitory effect of Origanum vulgare essential oil, thymol and carvacrol.

Biological properties of natural products are an important research target and essential oils (EO) from aromatic plants with antimicrobial properties are well documented. However, their uses are limited, and the mechanisms underlying their antibacterial activity are still not well known. Therefore, our objective was to evaluate the antibacterial activities of Origanum vulgare EO, thymol and carvacrol against Salmonella Enteritidis ATCC 13076 strain, particularly regarding the bacterial proteic profile, enzymatic activities and DNA synthesis. Bacterial expressed proteins were evaluated using an untreated assay control and treatments with sublethal concentrations of oregano EO, carvacrol and thymol. The same protein extracts were also assayed for oxidative stress and energy metabolism enzyme activities, as well as effect on DNA synthesis. Protein expression outcomes revealed by 2D-SDS-PAGE, from antimicrobial actions, showed a stress response with differential expressions of chaperones and cellular protein synthesis mediated by the bacterial signaling system. In addition, Salmonella used a similar mechanism in defense against oxidative stress, for its survival. Thus, the antibacterial inhibitory activity of EO was preferentially associated with the presence of thymol and there was interference in protein regulation as well as DNA synthesis affected by these compounds. SIGNIFICANCE: Antimicrobial activity of essential oils (EO) is already known. In this way, the understanding of how this activity occurs is a fundamental part to provide the practical and rational use of these substances. In the current scenario, where the emergence of resistant bacteria or even multiresistant bacteria against conventional antimicrobials, the search for alternatives becomes essential, since the discovery of new inhibitory substances does not occur at the same speed. The anti-Salmonella action allied to the knowledge about the biological processes affected by O. vulgare EO contribute to these bioactive compounds being effectively used as agents in the safety and shelf life of food in a future product, packaging or process where the antibacterial activity is safe and best used.

Vestibular function in severe GH deficiency due to an inactivating mutation in the GH-releasing hormone receptor gene.

PURPOSE: Body balance involves the vestibular, visual, and proprioceptive systems. IGF-I is a GH-dependent key factor in the development and postnatal differentiation of the inner ear in mice and men, but its role in the vestibular function in adult humans is unknown. We have previously described a cohort of individuals with severe isolated GH deficiency (IGHD) caused by a mutation in the GHRH receptor (GHRHR) gene. These individuals complain of dizziness, exhibit mild sensorineural loss, but have normal postural balance, without increase in falls risk. The aim of this study was to evaluate their vestibular function. METHODS: We performed physical examination (clinical head impulse and Fukuda dynamic stepping test), oculomotor (saccadic eye movements, spontaneous, semi-spontaneous and opotokinetic nystagmus, and pendular tracking) and caloric stimulation (postcaloric reflex and ocular fixation index) tests, in 15 GH-naïve IGHD (seven males) and 15 controls (five males). RESULTS: IGHD subjects showed lower height and weight, with similar BMI to controls, and higher number of individuals with abnormal clinical head impulse test and abnormal oculomotor tests, namely the saccadic movements and the spontaneous nystagmus. There was a nonsignificant trend in abnormalities in the Fukuda stepping test and postcaloric reflex test. CONCLUSIONS: Adult untreated IGHD individuals have higher prevalence of moderate peripheral vestibular impairment, and of abnormal vestibular-ocular reflex.