Effect of estradiol and bisphenol A on human hepatoblastoma cell viability and telomerase activity

Sex hormones from environmental and physiological sources might play a major role in the pathogenesis of hepatoblastoma in children. This study investigated the effects of estradiol and bisphenol A on the proliferation and telomerase activity of human hepatoblastoma HepG2 cells. The cells were divided into 6 treatment groups: control, bisphenol A, estradiol, anti-estrogen ICI 182,780 (hereinafter ICI), bisphenol A+ICI, and estradiol+ICI. Cell proliferation was measured based on average absorbance using the Cell Counting-8 assay. The cell cycle distribution and apoptotic index were determined by flow cytometry. Telomerase activity was detected by polymerase chain reaction and a telomeric repeat amplification protocol assay. A higher cell density was observed in bisphenol A (P<0.01) and estradiol (P<0.05) groups compared with the control group. Cell numbers in S and G2/M phases after treatment for 48 h were higher (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h (P<0.05) were higher in these groups than in the control group. The cell density was also higher in bisphenol A+ICI (P<0.01) and estradiol+ICI (P<0.05) groups compared with the ICI group. Furthermore, cell numbers were increased in S and G2/M phases (P<0.05), while the apoptotic index was lower (P<0.05) and telomerase activities at 48 and 72 h were higher (P<0.05) in these groups than in the ICI group. Therefore, bisphenol A and estradiol promote HepG2 cell proliferation in vitro by inhibition of apoptosis and stimulation of telomerase activity via an estrogen receptor-dependent pathway.

Effect of microRNA-21 on the proliferation of human degenerated nucleus pulposus by targeting programmed cell death 4

This study aims to explore the effect of microRNA-21 (miR-21) on the proliferation of human degenerated nucleus pulposus (NP) by targeting programmed cell death 4 (PDCD4) tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD) patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimics group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4 siRNAs). Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05). The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001). In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05). These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05). MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.

Evaluation of process parameters in the industrial scale production of fish nuggets

This work reports the use of experimental design for the assessment of the effects of process parameters on the production of fish nuggets in an industrial scale environment. The effect of independent factors on the physicochemical and microbiological parameters was investigated through a full 24 experimental design. The studied factors included the temperature of fish fillet and pulp in the mixer, the temperature of the added fat, the temperature of water and the ratio of protein extraction time to emulsion time. The physicochemical analyses showed that the higher temperature of the pulp and fillet of fish, the lower the protein in the final product. Microbiological analyses revealed that the counting of Staphylococcus coagulase positive, total and thermo-tolerant coliforms were in accordance with the current legislation.

Microbial flora in organic honey samples of africanized honeybees from Parana river islands

The purpose of this study was to verify and compare the main contamination sources and the hygienic/sanitary conditions of organic honey samples of Apis mellifera from Parana River islands. Thirty-three (33) samples were analyzed between January 2005 and August 2006. Eleven (11) samples were collected by beekeepers and twenty-two (22) samples were collected and processed in accordance with ideal personal hygiene norms and good manufacturing practices. The samples underwent microbiological analysis in search of coliforms at 35 ºC and 45 ºC, as well as fungi enumeration analysis. As for fungi counting, the samples harvested by beekeepers showed values above the maximum established by Resolution nº 15/94 of Common Market Group - Mercosul. The results showed that secondary contamination sources are responsible for the reduction of organic honey quality.

Culture alternative medium for the growth of extreme halophilic bacteria in fish products

Halotolerant or halophilic (Archaeabacteria) microorganisms can be found in salted and ripening fish products that are not affected by salt. They can be moderate or extremely halophilic bacteria. The extremely halophilic bacteria require between 15-30% of NaCl for growth. The extremely halophilic archaeobacteria may be selectively isolated in different media. The aim of this work was to determine the effectiveness of the Salt-Agar-Milk medium, a medium modified in our laboratory through the addition of MgSO4 and KCl - named SAMm, and its effect on the bacterial growth by means of comparison with other media, with and without milk, determining time of incubation and counting. Two samples of salted fish from local fish salting factories and two laboratory strains were used. The factory samples were matured anchovy and anchovy fillets in oil, and the laboratory strains were: Haloarcula spp. (proteolytic) and Halococcus spp. (non-proteolytic). The following media were alternatively used for the isolation of extremely halophilic bacteria: IRAM; Formulation of Gibbons and collaborators, Cod Milk agar, and SAMm. IRAM and Gibbons were also used enriched with milk. In the SAMm medium, there were obtained count values similar or higher than the ones of the traditional media; besides the simplicity of its elaboration, the possibility to obtain positive results two or three days earlier also added to its benefit. Consequently, it can be considered an alternative to the media traditionally used for the studied halophilic bacteria.

Stability and microbiological quality of rice bran subjected to different heat treatments

Rice bran is a byproduct commonly used for animal feeding; however its nutritional value and potential application in human diet have attracted market interest. Its preservation for safe use is still a challenge, so the objective of this study was to determine the quality of commercially available rice bran samples subjected to different heat treatments (extruding, parboiling, toasting, and microwave oven heating) in order to promote stabilization during storage under room temperature. Rice bran samples were collected from two industries, and each treatment was divided in three parts, each corresponding to three repetitions. All samples were evaluated for moisture content, total microorganisms, mold and yeast counting, hydrolytic rancidity, and lipase activity during 90 days of storage. Most of the heat treatments, including domestic and thermoplastic extrusion, generated products which may be used for human consumption under the tested conditions in terms of physicochemical and microbiological quality. The domestic treatments were more efficient in eliminating microorganisms or keeping them within acceptable limits. The toasted rice bran showed satisfactory results in terms of moisture, hydrolytic rancidity control, and lipase activity.

Quality of eggs sold in different commercial establishments and the study of the conditions of storage

The objective of this study was to investigate the influence of storage conditions on the physicochemical (mass, albumen height, pH, and Haugh unit) and microbiological (Salmonella spp., determination of the Total and Thermotolerant Coliforms, and Counting of the Mesophilic Aerobic Bacteria) quality of eggs. In the first experiment, a questionnaire was applied, and 33 samples of eggs were collected for the Salmonella spp analysis. In the second experiment, the eggs were collected from supermarkets, open markets, and distributors for physicochemical analysis. In the third experiment, 175 eggs were collected from the distributor, packaged in cardboard boxes lined with plastic wrapping, stored at 5 °C and 28 °C, and the physicochemical and microbiological analyses were performed at 7, 14, and 21 days of storage. In the first experiment, 100% of the samples analyzed showed no Salmonella spp. In the second experiment, it was found that the values of physicochemical parameters were in agreement with those in the literature. However, in the third experiment, the physicochemical parameter results showed statistical difference during storage and temperatures studied. Salmonella spp. were found in the samples stored at room temperature and in the refrigerated samples. Mesophilic microorganisms with values ranging from <10 CFU g- 1 (estimated) to 8.0 × 10³ CFU g- 1 and coliforms to 4 NMP.g- 1 were also found, but the presence of E. coli was not confirmed.

Evaluation of Petrifilm™ system compared with traditional methodology in count of indicators of sanitary-hygienic quality and pathogenic microorganisms in sheep milk

The objective of this study was to evaluate the applicability of the Petrifilm™ plates to enumerate microbial groups in sheep milk. Samples of sheep milk (n = 30) were plated simultaneously, to enumerate mesophilic aerobes, total coliforms, lactic acid bacteria, Staphylococcus aureus and Escherichia coli, using convencional reference protocols and Petrifilm™ plates. The results were compared using McNemar's test, linear regression and ANOVA (p < 0,05). The results demonstrated good significant between conventional methodologies and Petrifilm™ plates. Further, the Petrifim™ STX for counting S. aureus had higher recoverability of bacteria compared with the conventional methodology. Based on the results obtained and in view of the ease and rapidity procedures results, Petrifim ™ plates may be considered as alternatives for microbiological testing in sheep milk.

Quality indicators and shelf life of red octopus (Octopus maya) in chilling storage

Abstract There are no precedents concerning the quality of Octopus maya during chilled storage. This study evaluated the shelf life of the red octopus in chilling storage (4oC) and the correlation of the sensory quality index with microbiological counting and the biochemical indicators (hypoxanthine, histamine and volatile amines). A total of 112 whole raw octopi (average weight of 896 g) were randomly selected from seven batches and exposed to 4°C for 18, 24, 48, 72, 84, 96, and 100 h. The histamine concentration (91.7%), followed by the counts of psychrotrophic bacteria (5.5%) and hypoxanthine (2.2%), were the predictors from the redundancy analysis that better explained the changes taking place during the chilling hours. After 72 h of chilling, the microbial count was determined to be log 4.7 CFU/g, and the octopus samples were classified as B quality (minor sensory quality defects) based on the sensory quality scale. Although the samples were not classified as unacceptable at 100 h of refrigeration by the sensory index, the level of histamine reached the defect action level (5 mg/100 g) as ruled by the International Food Safety Authorities. The shelf life of the red octopus in chilling storage was predicted to be 119 h.

Physiological response of cowpea seeds to salinity stress

This work aimed to evoluate physiological response of cowpea (Vigna unguiculata L. Walp) seeds submitted to salt stress. Seeds of cultivars 'Epace-10'; 'Canapu' and 'Pitiúba' of cowpea, were submitted to germination test in germinator at 25(0)C, in "germitest" papers imbibed in distilled water or in 0, 10, 50, 100 and 200mol m-3 NaCl solutions. At the first and second counting of the germination test, normal seedlings were accounted, weighted and dried, obtaining data for vigor, total germination, fresh matter weight and dry matter weight. The seedlings hypocotyls, root and total length were measured total proteins content in cotyledons were obtained from germinating seeds. The presence of salt at concentrations higher than 50mol m-3 NaCl affect the germination, seedlings growth and cotyledons total protein synthesis of all cowpea cultivars. The seeds of cultivar pitiúba were is more tolerant to salinity, than the cultivars Canapu and Epace-10.

The use of film coating on the performance of treated corn seed

The main objective of seed coating technology using polymers is to improve the physical, physiological and sanitary characteristics of seed performance. The objectives of the present study were to determine: the plantability of corn seeds treated with insecticide, fungicide and graphite, covered with a film coating; the dust retention on treated corn seeds; and the leaching of applied products on corn seeds covered by a film coating. Seed plantability was determined by counting the skips and double seeds; dust was determined by using fiberglass paper in mg.100 g-1 of seeds; and the leaching was determined by collecting the material leached in a 10 cm layer of sand after irrigation. The following conclusions were made: seeds covered with film coating effectively reduce skips and double seeds; film coating effectively reduces the formation of dust from the seeds; film coated seeds minimize the leaching of the insecticide applied in seed treatment; and there are differences in effectiveness related to film coating type and dosage.

Discerning Cultural Depictions and Mistakes in Kafe Piter, a Russian Language Textbook for Finnish Students

This thesis presents the results of an analysis of the content in the series of Russian textbooks Kafe Piter, which is widely used in Finnish educational institutions for adult learners at the time that the research is conducted. The purpose of this study is to determine and describe how a textbook may purvey an image of a foreign country (in this case, Russia). Mixed-methods research with a focus on the qualitative content analysis of Kafe Piter is performed. The guidelines for textbook evaluation of cultural content proposed by Byram (1993) are used in this study as the basis for creating a qualitative analysis checklist, which is adopted according to the needs of the current research. The selection of the categories in the checklist is based on major themes where direct statements about Russia, Russian people and culture appear in the textbook. The cultural content and the way in which it is presented in Kafe Piter are also compared to the intercultural competence objectives of the Common European Framework of Reference for Languages. Because the textbook was not written by a native Russian speaker, it was also important to investigate the types of mistakes found in the books. A simple quantitative analysis in the form of descriptive statistics was done, which consisted of counting the mistakes and inaccuracies in Kafe Piter. The mistakes were categorized into several different groups: factual or cultural, lexicosemantic, grammatical, spelling and punctuation mistakes. Based on the results, the cultural content of Kafe Piter provides a rich variety of cultural information that allows for a good understanding of the Russian language and Russian culture. A sufficient number of cross-cultural elements also appear in the textbook, including cultural images and information describing and comparing Russian and Finnish ways of life. Based on the cultural topics covered in Kafe Piter, we conclude that the textbook is in line with the intercultural competence objectives set out in the Common European Framework of Reference for Languages. The results of the study also make it clear that a thorough proofreading of Kafe Piter is needed in order to correct mistakes - more than 130 cultural and linguistic mistakes and inaccuracies appear in the textbook.

Using stimulus equivalence to teach monetary skills to school-age children with autism

The present study evaluated the use of stimulus equivalence in teaching monetary skills to school aged children with autism. An AB within-subject design with periodic probes was used. At pretest, three participants demonstrated relation DA, an auditory-visual relation (matching dictated coin values to printed coin prices). Using a three-choice match-to-sample procedure, with a multi-component intervention package, these participants were taught two trained relations, BA (matching coins to printed prices) and CA (matching coin combinations to printed prices). Two participants achieved positive tests of equivalence, and the third participant demonstrated emergent performances with a symmetric and transitive relation. In addition, two participants were able to show generalization of learned skills with a parent, in a second naturalistic setting. The present research replicates and extends the results of previous studies by demonstrating that stimulus equivalence can be used to teach an adaptive skill to children with autism.

DNA polymerase activity in trophoblast giant cells in the mouse /

In the developing mouse embryo, the diploid trophectoderm is known to undergo a diploid to giant cell transformation. These cells arise by a process of endoreduplication, characterized by replication of the entire genome without subsequent mitosis or cell division, leading to polyploidy and the formation of giant nuclei. Studies of 13.5 day rat trophoblast derived from the parietal yolk sac have indicated a relatively low rate of DNA polymerase a activity, the noinnal eukaryotic replicase, in comparison to that of DNA polymerase g. These results have suggested that endoreduplication in trophoblast giant cells may not employ the normal replicase enzyme, DNA polymerase a. In order to determine whether a 'switch' from DNA polymerase to DNA polymerase is a necessary concomitant of the diploid to giant cell transformation, two distinct populations of trophoblast giant cells, the primary giant cell derived from the mural trophectoderm and the secondary giant cell derived from the polar trophoectoderm were used. These two populations of trophoblast giant cells can be obtained from the tissue outgrowths of 3.5da blastocysts and the extraembryonic ectoderm (EX) and ectoplacental cone (EPC) of 7.5 day embryos respectively. Tissue outgrowths were treated with aphidicolin, a specific reversible inhibitor of eukaryotic DNA polymerase a, on various days after explantation. The effect of aphidicolin treatment was assessed both qualitatively, using autoradiography and quantitatively by scintillation counting and Feulgen staining. 3 DNA synthesis was measured in control and treated cultures after a Hthymidine pulse. Scintillation counts of the embryo proper revealed that DNA synthesis was consistently inhibited by greater than 907. in the presence of aphidicolin. Inhibition of DNA synthesis in the EX and EPC varied between 81-957. and 82-987. respectively, indicating that most DNA synthesis was mediated by DNA polymerase a, but that a small but significant amount of residual synthesis was indicated. A qualitative approach was then applied to determine whether the apparent residual DNA synthesis was restricted to a subpopulation of giant cells or whether all giant cells displayed a low level of DNA synthesis. Autoradiographs of the ICM of blastocysts and the embryo proper of 7.5da embryos, which acted as diploid control population, was completely inhibited regardless of duration in explant culture. In contrast, primary trophoblast giant cells derived from blastocysts and secondary giant cells derived from the EX and EPC were observed to possess some heavily labelled cells after aphidicolin treatment. These results suggest that although DNA polymerase a is the primary replicating enzyme responsible for endoreduplication in mouse trophoblast giant cells, some nonactivity is also observed. A DNA polymerase assay employing tissue lysates of outgrown 7.5da embryo, EX and EPC tissues was used to attempt to confirm the presence of higher nonactivity in tissues possessing trophoblast giant cells. Employing a series of inhibitors of DNA polymerases, it would appear that DNA polymerase a is the major polymerase active in all tissues of the 7.5da mouse embryo. The nature of the putative residual DNA synthetic activity could not be unequivically determined in this study. Therefore, these results suggest that both primary and secondary trophoblast giant cells possess and use DNA polymerase a in endoreduplicative DNA synthesis. It would appear that the high levels of DNA polymerase g activity reported in trophoblast tissue derived from the 13.5 da rat yolk sac was not a general feature of all endoreduplication.

Gypsy moths (Lymantria dispar) in the Niagara Region : population density variation, introduction of an entomopathogenic fungus (Entomophaga maimaiga) and occurrence of nuclear polyhedrosis virus

The gypsy moth, Lymantria dispar, a major defoliator of broad leaf trees, was accidentally introduced into North America in 1869. Much interest has been generated regarding the potential of using natural pathogens for biological control of this insect. One of these pathogens, a highly specific fungus, Entomophaga maimaiga, was accredited with causing major epizootics in populations of gypsy moth across the north-eastern United States in 1989 and 1990 and is thought to be spreading northwards into Canada. This study examined gypsy moth population densities in the Niagara Region. The fungus, .E.. maimaiga, was artificially introduced into one site and the resulting mortality in host populations was noted over two years. The relationship between fungal mortality, host population density and occurrence of another pathogen, the nuclear polyhedrosis virus (NPV), was assessed. Gypsy moth population density was assessed by counting egg masses in 0.01 hectare (ha) study plots in six areas, namely Louth, Queenston, Niagara-on-the-Lake, Shorthills Provincial Park, Chippawa Creek and Willoughby Marsh. High variability in density was seen among sites. Willoughby Marsh and Chippawa Creek, the sites with the greatest variability, were selected for more intensive study. The pathogenicity of E. maimaiga was established in laboratory trials. Fungal-infected gypsy moth larvae were then released into experimental plots of varying host density in Willoughby Marsh in 1992. These larvae served as the inoculum to infect field larvae. Other larvae were injected with culture medium only and released into control plots also of varying host density. Later, field larvae were collected and assessed for the presence of .E.. maimaiga and NPV. A greater proportion of larvae were infected from experimental plots than from control plots indicating that the experimental augmentation had been successful. There was no relationship between host density and the proportion of infected larvae in either experimental or control plots. In 1992, 86% of larvae were positive for NPV. Presence and intensity of NPV infection was independent of fungal presence, plot type or interaction of these two factors. Sampling was carried out in the summer of 1993, the year after the introduction, to evaluate the persistence of the pathogen in the environment. Almost 50% of all larvae were infected with the fungus. There was no difference between control and experimental plots. Data collected from Willoughby Marsh indicated that there was no correlation between the proportion of larvae infected with the fungus and host population density in either experimental or control plots. About 10% of larvae collected from a nearby site, Chippawa Creek, were also positive for .E.. maimaiga suggesting that low levels of .E.. maimaiga probably occurred naturally in the area. In 1993, 9.6% of larvae were positive for NPV. Again, presence or absence of NPV infection was independent of fungal presence plot type or interaction of these two factors. In conclusion, gypsy moth population densities were highly variable between and within sites in the Niagara Region. The introduction of the pathogenic fungus, .E.. maimaiga, into Willoughby Marsh in 1992 was successful and the fungus was again evident in 1993. There was no evidence for existence of a relationship between fungal mortality and gypsy moth density or occurrence of NPV. The results from this study are discussed with respect to the use of .E.. maimaiga in gypsy moth management programs.