Identification of Treponema denticola in subgingival samples by PCR technology and its correlation with clinical diagnosis

Treponema denticola has been associated with gingivitis and chronic periodontitis. The aim of this study was to identify Treponema denticola in subgingival samples using PCR technology and to correlate it with clinical diagnosis of subjects. The study was carried out on seventy patients (20-84 years of age; mean age, 45.06 +/- 12.58) of which 22 individuals with no detectable gingivitis or periodontitis, 4 subjects with chronic gingivitis and 44 subjects with chronic periodontitis. Subgingival plaque samples were collected from five sites in each patient. DNA was extracted from the samples using QIAamp DNA Mini Kit (QIAGEN). Treponema denticola and other four periodontopathogens were found using multiplex polymerase chain reaction followed by a reverse hybridization. The relationship between clinical diagnoses and detection of Treponema denticola was determined with Fisher exact test. The results showed significant differences between diagnostic groups regarding subject proportion. Treponema denticola was detected in 2 out of 22 subjects with no detectable gingivitis or periodontitis, 2 out of 4 subjects with chronic gingivitis, and 40 out of 44 subjects with chronic periodontitis. Our findings suggest that Treponema denticola is closely connected to the initiation and progression of periodontal disease.     

Cereulide-producing strains of <Emphasis Type="Italic">Bacillus cereus</Emphasis> show diversity

Producers of cereulide, the emetic toxin of Bacillus cereus, are known to constitute a specific subset within this species. We investigated physiological and genetic properties of 24 strains of B. cereus including two high cereulide producers (600–1,800 ng cereulide mg⁻¹ wet weight biomass), seven average producers (180–600 ng cereulide mg⁻¹ wet weight biomass), four low cereulide producers (20–160 ng cereulide mg⁻¹ wet weight biomass) and 11 non-producers representing isolates from food, food poisoning, human gut and environment. The 13 cereulide producers possessed 16S rRNA gene sequences identical to each other and identical to that of B. anthracis strains Ames, Sterne from GenBank and strain NC 08234–02, but showed diversity in the adk gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three types of patterns), in tyrosin decomposition, haemolysis and lecithin hydrolysis (two phenotypes). The cereulide-producing isolates from the human gut represented two ribopatterns of which one was novel to cereulide-producing B. cereus and two phenotypes. We conclude that the cereulide-producing B. cereus are genetically and biochemically more diverse than hitherto thought.     

The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.     

High-throughput phagocytosis assay utilizing a pH-sensitive fluorescent dye

We describe a development of a novel high-throughput phagocytosis assay based on a pH-sensitive cyanine dye, CypHer5E, which is maximally fluorescent in an acidic environment. This dye is ideally suited for the study of phagocytosis because of the acidic conditions generated in the intracellular phagocytic vesicles after particle uptake. Use of CypHer5E-labeled particles results in greatly reduced background from noninternalized particles and makes the assay more robust. Additionally, CypHer5E-labeled particles are resistant to fluorescence quenching observed in the aggressive and acidic environment of the phagosome with traditional dyes. The CypHer5E-based assay has been shown to work reliably in a variety of cell types, including primary human monocytes, primary human dendritic cells, primary human endothelial cells, human monocytic THP-1 cell line, and human/mouse hybrid macrophage cell line WBC264-9C. Inhibition of CypHer5E bead uptake by cytochalasin D was studied, and the 50% inhibition concentration (IC50) was determined. The assay was performed in 96- and 384-well formats, and it is appropriate for high-throughput cellular screening of processes and compounds affecting phagocytosis. The CypHer5E phagocytosis assay is superior to existing protocols because it allows easy distinction of true phagocytosis from particle adherence and can be used in microscopy-based measurement of phagocytosis.     

Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid

Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.     

Identification of four Treponema species in subgingival samples by nested-PCR and their correlation with clinical diagnosis

The relationship between different species of oral Treponemas and inflammation in periodontal disease progression is complex. The purpose of this study was to analyze and compare the subgingival plaque samples collected from periodontally healthy subjects and from chronic gingivitis and periodontitis patients in order to detect the presence of T. denticola, T. pectinovorum, T. socranskii and T. vincentii using nested-PCR technology. After DNA extraction from the samples using QIAmp DNA Mini Kit (QIAGEN, the four Treponema species were determined with nested-polymerase chain reaction which requires two sets of primers to amplify a specific DNA fragment in two separate runs of PCR. Pearson chi-square was implemented to compare the three groups as to the presence of four Treponema species. Results of this investigation showed significant differences between groups regarding subject proportion of T. denticola, T. socranskii, T. pectinovorum, T. vincentii, with a higher percentage of patients from associated-disease groups of patients harboring these four species than healthy subjects. These differences were more pronounced in presence of Treponema denticola and Treponema socranskii. Our findings suggest that Treponema denticola and Treponema socranskii concurrent presence indicate more accurately the association with chronic gingivitis and periodontitis.     

Application of Microencapsulated Synbiotics in Fruit-Based Beverages

In the last years, demand for functional products containing both prebiotics and probiotics (known as synbiotic) has increased, which stimulated their incorporation into other food matrices than milk-based ones. Synbiotics improve gut functionality as well as respond to the increasing demand of consumers who have become aware of the health benefits of a proper diet. The most important criterion for preserving consumer acceptance in such products is maintaining the minimum viability and activity of probiotics from the beginning of production to the end of shelf-life. For their viability, fixation and multiplying within the host, several solutions have been proposed including the fortification with prebiotics and microencapsulation of prebiotics along with probiotics. The challenge of microencapsulation is to protect the probiotic cells in foods that are not usually considered their vehicle, such as fruit matrices. It is generally known that different prebiotics may exert different degrees of protection on the entrapped bacteria cells. For food products, such as fruit beverages, few works exist that investigate the functionality of synbiotic microcapsules in protecting the survivability of probiotic cells during processing and storage. This article provides an overview of this novel trend based on a review of relevant literature. The article summarizes the synbiotic concept, challenges for synbiotic formulation in fruit beverages, and future perspectives.

     

Antioxidant activity,acetylcholinesterase and tyrosinase inhibitory potential of Pulmonaria officinalis and Centarium umbellatum extracts

In this study several investigations and tests were performed to determine the antioxidant activity and the acetylcholinesterase and tyrosinase inhibitory potential of Pulmonaria officinalis and Centarium umbellatum aqueous extracts (10% mass) and ethanolic extracts (10% mass and 70% ethanol), respectively. Moreover, for each type of the prepared extracts of P. officinalis and of C. umbellatum the content in the biologically active compounds – polyphenols, flavones and proanthocyanidins was determined. The antioxidant activity was assessed using two methods, namely the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and reducing power assay. The analyzed plant extracts showed a high acetylcholinesterase and tyrosinase inhibitory activity in the range of 72.24–94.24% (at the highest used dose – 3 mg/mL), 66.96% and 94.03% (at 3 mg/mL), respectively correlated with a high DPPH radical inhibition – 70.29–84.9% (at 3 mg/mL). These medicinal plants could provide a potential natural source of bioactive compounds and could be beneficial to the human health, especially in the neurodegenerative disorders and as sources of natural antioxidants in food industry.