Oral health status of Egyptian children with lysosomal storage diseases: An evaluation of dental indices, salivary cytokines level, and bacterial bioburden.

BACKGROUND: Lysosomal storage diseases (LSDs), a group of inborn errors of metabolism, include various subtypes, for example, mucopolysaccharidosis (MPS) and Gaucher disease (GD). Besides the physical/mental disabilities, they suffer from several oral deteriorations. AIM: To evaluate the oral health status of Egyptian children with LSD. DESIGN: Thirty LSD children and thirty non-LSD children were enrolled for this study according to the inclusion and exclusion criteria. Dental indices were used to assess caries prevalence and periodontal status. Saliva samples were collected from all enrolled children to estimate interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and protein levels as well as Streptococcus mutans and Lactobacilli colony counts. RESULTS: Children with MPS and GD showed non-significant differences in decayed, missing, or filled teeth (DMFT) scores (p = .115). Scores of dmft showed a significant increase in MPS, but not in GD children (p = .020, p = .127). Children with LSD showed significantly increased Modified Gingival Index (MGI), Plaque Index (PI), Oral Hygiene Index (OHI-s) scores (p < .001) and salivary IL-6 and TNF-α (p = .007, p = .001, p < .0001, p = .002, respectively) and salivary total proteins (p = .001) levels. Unexpectedly, non-significant differences were observed in salivary Streptococcus mutans or Lactobacilli counts in children with MPS and GD (p = .058, p = .420, p = .502, p = .053, respectively). CONCLUSION: To our knowledge, this is the first article that evaluates Egyptian children with LSD. We demonstrated high caries prevalence in primary teeth, not permanent teeth, in children with MPS and poor gingival/hygiene status in children with MPS and GD, which triggered a state of inflammation. The daily supplement intake prevented oral bacterial growth. The most probable cause of oral alterations is decreased salivary flow rate, as deduced from a significantly increased salivary protein.

Improved production of Bacillus subtilis cholesterol oxidase by optimization of process parameters using response surface methodology.

BACKGROUND: Cholesterol oxidase has numerous biomedical and industrial applications. In the current study, a new bacterial strain was isolated from sewage and was selected for its high potency for cholesterol degradation (%) and production of high cholesterol oxidase activity (U/OD600). RESULTS: Based on the sequence of 16S rRNA gene, the bacterium was identified as Bacillus subtilis. The fermentation conditions affecting cholesterol degradation (%) and the activity of cholesterol oxidase (U/OD600) of B. subtilis were optimized through fractional factorial design (FFD) and response surface methodology (RSM). According to this sequential optimization approach, 80.152% cholesterol degradation was achieved by setting the concentrations of cholesterol, inoculum size, and magnesium sulphate at 0.05 g/l, 6%, and 0.05 g/l, respectively. Moreover, 85.461 U of cholesterol oxidase/OD600 were attained by adjusting the fermentation conditions at initial pH, 6; volume of the fermentation medium, 15 ml/flask; and concentration of cholesterol, 0.05 g/l. The optimization process improved cholesterol degradation (%) and the activity of cholesterol oxidase (U/OD600) by 139% and 154%, respectively. No cholesterol was detected in the spectroscopic analysis of the optimized fermented medium via gas chromatography-mass spectroscopy (GC-MS). CONCLUSION: The current study provides principal information for the development of efficient production of cholesterol oxidase by B. subtilis that could be used in various applications.

Biosynthesis of Silver Nanoparticles by Aspergillus terreus: Characterization, Optimization, and Biological Activities.

In this study, mycelial filtrate of Aspergillus terreus BA6 was used to reduce AgNO3 to form silver nanoparticles (AgNPs). The effect of seven independent variables on the diameter of AgNPs was studied by applying design of experiments (DOE). At optimal conditions, the diameter of AgNPs was reduced by approximately 26.7% compared to the basal culture condition and AgNO3 concentration was found to be the most significant factor affecting the diameter of AgNPs. A. terreus nano-Ag was characterized using UV-visible spectroscopy, transmission electron microscopy, energy dispersive X-ray (EDX), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and Zeta potential. The maximum UV absorption was obtained at 420 nm and the microscopic results showed particles with narrow size distribution ranging from 7 to 23 nm. XRD pattern of AgNPs revealed four diffraction peaks of metallic silver and the EDX spectrum showed a strong signal attributed to Ag nano-crystals. AgNPs mycofabricated by A. terreus showed potent minimum inhibitory concentration (MIC) and broad minimum bactericidal/fungicidal concentration (MBC/MFC) against 12 reference microorganisms. The MIC and MBC/MFC values of AgNPs were 0.312 to 1.25 µg/ml and 0.625 to 10 µg/ml, respectively. Nevertheless, AgNPs did not demonstrate any antagonistic activity against Coxsackie B virus. The in vitro cytotoxicity of the mycosynthesized AgNPs showed significant antitumor activity against adenocarcinoma epithelial cells from human breast cancer (Mcf-7) cell line with an inhibitory concentration (IC50) of 87.5 µg/ml.

The Inhibitory Effect of Wheat Husks Addition on Aflatoxins Production by Aspergillus flavus in Liquid Culture With Various Wheat Compositions as Carbon Sources.

Wheat may be infected by the aflatoxigenic mold Aspergillus flavus during pre- and post-harvest activities. Control strategies reported to manage aflatoxin contamination of wheat are expensive and require extensive testing to verify the absence of toxic secondary metabolites or newly formed compounds. The objective of this study was to develop an in vitro new control strategy based on assessing the influence of wheat husks on aflatoxin production by A. flavus in liquid culture. The results showed that aflatoxin production is significantly influenced by the existence of husks in the wheat forms used as carbon substrates according to the following order: full wheat grains < half-crushed wheat grains < wheat flour 82% < wheat flour 72%. By applying a fractional factorial design and a response surface methodology, maximum aflatoxin production (2.567 ng/mg) was predicted when wheat flour 72% (39 g/l) as a carbon source, yeast extract (5 g/l), and a 75-ml medium volume/250 ml flask were utilized. At this optimized condition, after addition of wheat husk extract, the growth and synthesis of aflatoxins of A. flavus were repressed by 74.85 and 98.72%, respectively. This finding paves the way to examine the antifungal potential of wheat husk constituents and to compare their efficacy with thyme, cinnamon, sweet basil, and coriander essential oils, which possess antimycotic activities. Accordingly, the wheat husk component SiO2 showed the highest growth inhibition (67.04%) and reduction of A. flavus aflatoxins (82.67%). These results are comparable to those obtained from various examined antimycotic essential oils.

Kinetic studies on the expression of alginate and extracellular proteins by Pseudomonas aeruginosa FRD1 and PAO1.

Pseudomonas aeruginosa is characterized by its capability to produce extracellular virulence proteins and to establish biofilm-based infections that do not respond easily to conventional treatments. However, the physiological conditions that decrease the fitness of such a persistent pathogen would assist the host to defend itself and reduce the infection prevalence. Therefore, developing treatments against P. aeruginosa requires a quantitative understanding of the relationship between bacterial growth kinetics and secretion of alginate and proteins, in addition to the ecological factors that control their synthesis. For this purpose, we examined various environmental factors that affect the specific product yield coefficients (expressed as g product/OD600) of alginate and extracellular proteins using a mucoid (FRD1) and a non-mucoid (PAO1) clinical isolate of P. aeruginosa, respectively. The results suggested magnesium sulfate, trace elements and hydrogen peroxide as significant variables that positively affect alginate synthesis by the FRD1 cells. However, the production of extracellular proteins by PAO1 was negatively affected by the concentration of ferrous sulfate. For understanding the kinetics of expressing alginate and extracellular proteins by the cells, a well-controlled 5 L tank bioreactor was used. The results suggested that under the bioreactor controlled conditions, both alginate and extracellular proteins are expressed parallel to biomass increase in the cells of P. aeruginosa.

Production of di-(2-ethylhexyl) phthalate by Bacillus subtilis AD35: Isolation, purification, characterization and biological activities.

The emergence of extensive antibiotics resistant bacteria increased the demands for finding out new sources of antimicrobial agents. Marine niches were reported to be rich in many competent producers of significant bioactive compounds. On the course of screening program for new antimicrobials, a Bacillus strain was isolated from Alexandria sea shores, Egypt. According to the morphological, cultural and biochemical characteristics, 16S rRNA sequence analysis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), the strain was identified as Bacillus subtilis and designated as B. subtilis AD35. One phthalate derivative namely Di-(2-ethylhexyl) phthalate (DEHP) was purified from the crude extract of B. subtilis AD35 by high performance liquid chromatography (HPLC) and the structural elucidation of this compound was confirmed on the basis of gas chromatography-mass spectroscopy (GC-MS), Fourier infrared spectroscopy (FT-IR) and UV spectrum. The results of MIC of the purified DEHP were as follow: 16 µg/ml (Salmonella typhimurium), 32 µg/ml (Methicillin resistant Staphylococcus aureus, MRSA), 0.25 µg/ml (Listeria monocytogenes), 0.5 µg/ml (Aeromonas hydrophila), 8 µg/ml (Staphylococcus aureus), 4 µg/ml (Staphylococcus epidermidis), 4 µg/ml (Escherichia coli), and 8 µg/ml (Pseudomonas aeruginosa). DEHP produced by B. subtilis AD35 up to a concentration of 2500 µg/ml exhibited no cytotoxic effect against normal Vero cells. In addition, it did not show an antiviral activity against HAV or a significant growth inhibitory effect toward human colorectal adenocarcinoma and human mammary gland adenocarcinoma cell-lines.

Expression of extracellular polysaccharides and proteins by clinical isolates of Pseudomonas aeruginosa in response to environmental conditions.

The opportunistic pathogen Pseudomonas aeruginosa causes chronic respiratory infections in patients with cystic fibrosis (CF). Persistence of this bacterium is attributed to its ability to form biofilms which rely on an extracellular polymeric substance matrix. Extracellular polysaccharides (EPS) and secreted proteins are key matrix components of P. aeruginosa biofilms. Recently, nebulized magnesium sulfate has been reported as a significant bronchodilator for asthmatic patients including CF. However, the impact of magnesium sulfate on the virulence effect of P. aeruginosa is lacking. In this report, we investigated the influence of magnesium sulfate and other environmental factors on the synthesis of alginate and secretion of proteins by a mucoid and a non-mucoid strain of P. aeruginosa, respectively. By applying the Plackett-Burman and Box-Behnken experimental designs, we found that phosphates (6.0 g/l), ammonium sulfate (4.0 g/l), and trace elements (0.6 mg/l) markedly supported alginate production by the mucoid strain. However, ferrous sulfate (0.3 mg/l), magnesium sulfate (0.02 g/l), and phosphates (6.0 g/l) reinforced the secretion of proteins by the non-mucoid strain.

Production of a thermostable uricase by a novel Bacillus thermocatenulatus strain.

A novel uricase-producing bacterium was identified based on its 16S rRNA sequence as Bacillus thermocatenulatus. The kinetic constants for this uricase, determined with uric acid as the substrate, were a V(max) of 0.99U/ml of enzyme and a K(m) of 0.25mM. After heat treatment at 75 degrees C for 45min, the uricase retained about 100% of its initial activity. The uric acid showed to be an inducer for uricase production. The effects of different factors on the enzyme production were studied. Pretreated cane molasses and corn steep liquor were the most promising carbon and nitrogen sources, respectively. When the strain was cultured at 30 degrees C at pH 7.0 for 30-36h, the uricase activity peaked at 1.25U/ml.

Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs.

In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of citric acid in submerged culture. For screening of fermentation medium composition significantly influencing citric acid production, the two-level Plackett-Burman design was used. Under our experimental conditions, beet molasses and corn steep liquor were found to be the major factors of the acid production. A near optimum medium formulation was obtained using this method with increased citric acid yield by five-folds. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, the three-level Box-Behnken design was applied. A polynomial model was created to correlate the relationship between the three variables (beet molasses, corn steep liquor and inoculum concentration) and citric acid yield. Estimated optimum composition for the production of citric acid is as follows pretreated beet molasses, 240.1g/l; corn steep liquor, 10.5g/l; and spores concentration, 10(8)spores/ml. The optimum citric acid yield was 87.81% which is 14 times than the basal medium. The five level central composite design was used for outlining the optimum values of the fermentation factors initial pH, aeration rate and temperature on citric acid production. Estimated optimum values for the production of citric acid are as follows initial pH 4.0; aeration rate, 6500ml/min and fermentation temperature, 31.5 degrees C.

Citric acid production by a novel Aspergillus niger isolate: I. Mutagenesis and cost reduction studies.

Ultraviolet-irradiation (UV), ethyl methane sulfonate (EMS) and acridine orange (AO) were used to induce citric acid overproduction mutations in Aspergillus niger UMIP 2564. Among 15, eight of the mutant derivatives, were improved with respect to citric acid production from sucrose in batch cultures. Maximum product yield (60.25%) was recorded by W5, a stable UV mutant, with approximately 3.2-fold increase when compared to the parental wild type strain. In terms of the kinetic parameters for batch fermentation processes, the mutation doubled the specific substrate uptake rate and achieved 4.5- and 7.5-fold improvements in citric acid productivity and specific productivity, respectively. For reduction of the fermentation medium cost, corn steep liquor and calcium phosphate pre-treated beet molasses were successfully used as substituents of nitrogen and carbon sources in the growth medium, respectively. These medium substitutions resulted in a W5 citric acid fermentation culture with a product yield of 74.56%.

The utilization of beet molasses as a novel carbon source for cephalosporin C production by Acremonium chrysogenum: Optimization of process parameters through statistical experimental designs.

In this work, cephalosporin C (CPC) production on pilot scale fermenters of 600l capacity with 350l working volume by Acremonium chrysogenum EMCC 904 was performed. The effects of fermentation medium composition, inoculum concentration, initial pH and aeration rate on CPC production by A. chrysogenum strain was investigated by using response surface methodology (RSM). The Plackett-Burman design which involves two concentrations of each nutrient was effective in searching for the major medium components promoting CPC production. Under our experimental conditions; Soya oil, beet molasses and corn steep liquor were found to be the major factors contributing to the antibiotic production. Subsequently, a Box-Behnken design was used for outlining the concentration of the most effective medium constituents. Estimated optimum composition for the production of CPC was as follows: soya oil, 40g/l; beet molasses, 180g/l; and corn steep liquor, 330g/l. The central composite design was used for outlining the optimum values of the fermentation parameters. Estimated optimum values for the production of CPC are as follows: inoculum level, 10(5.5)spores/ml; initial pH, 4.3; and aeration rate, 9364ml/min.