In Vitro Antimicrobial Activity and Downregulation of Virulence Gene Expression on <Emphasis Type="Italic">Helicobacter pylori</Emphasis> by Reuterin

Helicobacter pylori is an infectious agent commonly associated with gastrointestinal diseases. The use of probiotics to treat this infection has been documented, however, their potential antimicrobial metabolites have not yet been investigated. In the present study, the effect of reuterin produced by Lactobacillus reuteri on H. pylori growth and virulence gene expression was evaluated. It was observed that reuterin caused significant (P < 0.05) H. pylori growth inhibition at concentrations from 0.08 to 20.48 mM, with minimal inhibitory concentrations (MICs) of 20.48 mM for H. pylori ATCC700824 and 10.24 mM for H. pylori ATCC43504. In a reuterin bacterial killing assay, it was observed that half of the MIC value for H. pylori (ATCC700824) significantly (P < 0.01) reduced colony numbers from 5.65 ± 0.35 to 3.78 ± 0.35 Log₁₀ CFU/mL after 12 h of treatment and then increased them to 5.25 ± 0.23 Log₁₀ CFU/mL at 24 h; at its MIC value (20.48 mM), reuterin abrogated (P < 0.01) H. pylori (ATCC700824) growth after 20 h of culture. In addition, reuterin significantly (P < 0.01) reduced H. pylori (ATCC 43504) colony numbers from 5.65 ± 0.35 to 4.1 ± 0.12 Log10 CFU/mL from 12 to 24 h of treatment and abrogated its growth at its MIC value (10.24 mM), after 20 h of treatment. Reuterin did not alter normal human gastric Hs738.St/Int cell viability at the concentrations tested for H. pylori strains. Furthermore, 10 μM reuterin was shown to significantly (P < 0.01) reduce mRNA relative expression levels of H. pylori virulence genes vacA and flaA at 3 h post-treatment, whose effect was higher at 6 h post-treatment, as measured by RT-qPCR. The observed direct antimicrobial effect and the downregulation of expression of virulence genes on H. pylori by reuterin may contribute to the understanding of the mechanisms of action of probiotics against H. pylori.     

Membrane lipids and ethanol tolerance in the mouse. The influence of dietary fatty acid composition

Mice of the TO Swiss strain received diets containing different amounts of saturated or unsaturated fat throughout life. These diets produced characteristic changes in cardiac phospholipid fatty acid composition, but produced no significant differences in fatty acid composition of phospholipids from a crude membrane fraction of brain. When littermates of these animals were exposed to ethanol vapour in an inhalation chamber it was observed that mice which had received a diet high in saturated fat lost the righting reflex at an estimated concentration of ethanol in blood higher than that required for mice receiving a control diet, or a diet rich in polyunsaturated fat. Analysis of the brain membrane fraction from those animals which had received ethanol revealed that mice receiving the highly saturated fat diet now had a significantly greater proportion of saturated fatty acids in brain membrane phospholipids. These results are discussed in relation to the hypothesis that brain membrane lipid composition may influence the behavioural response to ethanol.     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Reproductive traits of the freshwater oyster Etheria elliptica (Bivalvia: Etheriidae) in the Pendjari River,Benin: implications for conservation

The African freshwater oyster Etheria elliptica, which is of great economic importance throughout the continent, is facing overharvesting in many fisheries in West Africa. Its reproductive traits (sex ratio, size at sexual maturity, oocyte diameter and fecundity) were studied at four stations located along the Pendjari River, northern Benin, in April 2013. Histological techniques were implemented to identify sex and gonad development stages. Oocyte sizes were measured based on the histological images and mean oocyte diameter was 38 µm. Fecundity, estimated by counting the developing eggs, averaged 106 724 eggs and increased significantly with shell size. The average sex ratio was approximately 1:1. Hermaphrodites were rare. Males reached sexual maturity at 57.6 mm dorsoventral height, earlier than females at 64.7 mm. The majority of the oyster specimens were in ripe-spawning stages, indicating that reproductive activities partly took place towards the end of the dry season. Both mature and immature individuals were subjected to harvesting at all the investigated fishing sites. The mean size of exploited oysters was 60.6 mm, lower than the size of females at maturity. Management strategies must enforce the minimum size of 65 mm required for harvesting, particularly in open access sites where juveniles are mostly harvested.     

A new strategy for the decellularization of whole organs by hydrostatic pressure

Tissue engineering has been able to develop novel decellularization-recellularization techniques, which facilitates the research for the generation of functional organs. This is based in the initial obtention of the organ's extracellular matrix (ECM). Therefore, any improvement in the decellularization process would have a positive impact in the results of the recellularization process. Nevertheless, commonly the methods and equipment employed for this process are expensive and thus limit the access of this technique to various research groups globally. To develop a decellularization technique with the exclusive use of hydrostatic pressure of detergent solutions, to have an easily accessible and low-cost technique that meets the basic requirements of acellularity and functionality of the ECM. This experimental study was performed in 10 male Wistar rats, obtaining the liver to carry out serial washes, with 1%, 2%, and 3% Triton X-100 solutions and 0.1% SDS. The washes were performed by using a gravity perfusion system (GPS), which assured us a continuous hydrostatic pressure of 7.5 mmHg. The obtained ECM was processed using stains and immunostaining to determine the residual cell content and preservation of its components. The staining showed a removal of cellular and nuclear components of approximately 97% of the acellular ECM, with an adequate three-dimensional pattern of collagen and proteoglycans. Furthermore, the acellular ECM allowed the viability of a primary hepatocyte culture. The use of the GPS decellularization technique allowed us to obtain an acellular and functional ECM, drastically reducing experimentation costs.