Effect of sample storage time on detection of hybridization signals in Checkerboard DNA-DNA hybridization

Long-term sample storage can affect the intensity of the hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the hybridization signals of 13 bacterial species detected by the Checkerboard DNA-DNA hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n?= 24) and (2) after storage at -20?°C for 6 months (n?= 24), (3) for 12 months (n?= 24), and (4) for 24 months (n?= 24). The intensity of hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p?< 0.001). No differences were found between groups 1 and 2 (p?> 0.05). The Checkerboard DNA-DNA hybridization method was suitable to detect hybridization signals from all groups evaluated, and the intensity of signals decreased significantly after long periods of sample storage.     

Investigation of the radiation effects on Brown Rot disease of Golden Delicious apples,inoculated with the fungus Monilinia fructigena

The effects of 0, 1.5 and 3 KGy gamma-irradiation on Brown Rot disease affecting Golden Delicious apples are investigated. The apples were artificially inoculated with Monilinia fructigena fungus before and after irradiation. Results show clearly that radiation induces a delay in fungus development, even when it is not directly affected. The effect of radiation on the apples' substrate seems to be considerable, being the main factor affecting fungal growth. Radiation doses of 1.5 and 3 KGy do not seem to produce strong differences in the fungal growth. Irradiation, however, is very effective, producing important changes of practical interest for apples infected before or after irradiation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Discrimination of gamma-irradiated and nonirradiated Vibrio vulnificus by using real-time polymerase chain reaction

Aims: To develop a PCR strategy for Vibrio vulnificus in irradiated foods. Methods and Results: Real‐time PCR was used to discriminate between DNA from γ‐irradiated and nonirradiated cells. γ‐Irradiation at 1·08 KGy and above of cell suspensions containing 1 × 10⁶ CFU ml^?1 resulted in 0 CFU ml^?1. Real‐time PCR was able to detect 86·6% destruction by 1·08 KGy, while ethidium bromide monoazide (EMA) real‐time PCR was able to detect 93·2% destruction at this dose. With 3·0 and 5·0 KGy, EMA real‐time PCR was able to detect 99·3% and 100% destruction, respectively. Conclusions: The inability to detect via PCR extensively degraded DNA resulting from γ‐irradiation can be taken as evidence of cell death. The increased ability of EMA to further reduce the detectable number of target sequences via PCR, with DNA from cells exposed to increased doses of γ‐irradiation, can be considered to reflect the accompanying increase in membrane damage which allows EMA to penetrate the cells. Significance and impact of the study: This is the first study that has made use of the PCR to discriminate between γ‐irradiated and nonirradiated bacterial cells and has led to insights regarding the ability of the PCR to discriminate between nonirradiated and γ‐irradiation destroyed cells.     

三硝基甲苯致晶状体上皮细胞DNA损伤作用的研究

本文通过出生后７天和２４个月两组大鼠晶状体与２０％三硝基甲苯（ＴＮＴ）甘油：水（８：２）混悬液体外培养,培养箱通５％ＣＯ２,恒温３７℃,观察ＴＮＴ对晶状体上皮细胞ＤＮＡ的损伤程度,结果以反映单链ＤＮＡ断裂强度的Ｆ６３０／Ｆ５３０比值表示。ＨＰＬＣ分析发现经体外ＴＮＴ孵育的晶状体内含有ＴＮＴ及其代谢产物;当共同培养９６ｈ后,Ｆ６４０／Ｆ５３０比值随着ＴＮＴ剂量增加而增大,达到４０μＬ（１１．７４μｍｏｌ／Ｌ）时趋向平稳;当用ＴＮＴ浓度为４．４０μｍｏｌ／Ｌ的相同剂量培养时,４８小时后单链ＤＮＡ断裂最为严重Ｆ６３０／Ｆ５３０比值为１．５０,明显高子正常对照组（Ｐ＜０．０１）;幼鼠单链ＤＮＡ断裂程度显著高于老龄鼠（Ｐ＜０．０１）。     

1H NMR and MVA metabolomic profiles of urines from piglets fed with boluses contaminated with a mixture of five mycotoxins

Metabolic profile of urine from piglets administered with single boluses contaminated with mycotoxin mixture (deoxynivalenol, aflatoxin B₁, fumonisin B₁, zearalenone, and ochratoxin A) were studied by ¹H NMR spectroscopy and chemometrics (PCA, PLS-DA, and OPLS-DA). The mycotoxin levels were close to the established maximum and guidance levels for animal feed (2003/100/EC and 2006/576/EC). Urine samples were obtained from four groups of four piglets before (control, C) or within 24 h (treated, T) after receiving a contaminated boluses with increasing doses of mycotoxins (boluses 1–4). For the two highest dose groups, the urines were collected also after one week of wash out (W). For the two lowest doses groups no significant differences between the C and T samples were observed. By contrast, for the two highest doses groups the T urines separated from the controls for a higher relative content of creatinine, p-cresol glucuronide and phenyl acetyl glycine and lower concentration of betaine and TMAO. Interestingly, a similar profile was found for both W and T urines suggesting, at least for the highest doses used, serious alteration after a single bolus of mycotoxin mixture.     

Effect of gamma irradiation on elimination of aflatoxins produced by apple mycoflora in apple fruits

The influence of ionizing radiation on the growth of apple mycoflora and the aflatoxins production was investigated. Four strains of fungal isolates, Aspergillus flavus, A. fumigatuis, A. niger, Penicillium expansum, were used. Four doses of gamma radiation were applied (0.5, 1.0, 1.5 and 2.0 KGy). The elimination of aflatoxins under selected conditions was detected by using HPLC techniques. Doses of 0.5 KGy stimulated the production of aflatoxins by all fungi tested while 1.0-2.0 KGy reduced the aflatoxins production. The studies were also carried out to investigate the effect of pre- and post-inoculation of both irradiated and non-irradiated fungal spores on irradiated and non-irradiated apples. The results showed the necessity of irradiation of fresh apple fruits followed by good preservation conditions. The results showed also that the effect of radiation is influences on the apple mycoflora producing aflatoxins and not the apple itself.     

Oxidative Tissue Response Promoted by 5-Aminolevulinic Acid Promptly Induces the Increase of Plasma Antioxidant Capacity

The heme precursor 5-aminolevulinic acid (ALA), acting as a prooxidant, has been proposed to underlie the clinical manifestations of various porphyric disorders. Accordingly, ALA-generated oxyradicals where shown to cause oxidative lesions in biomolecules and isolated cell organelles and to release iron from ferritin. In rats, administered ALA triggered oxidative stress in liver, brain and red muscles. We now study the correlation between the plasma antioxidant capacity and tissue oxidative damage, after acute (one and two doses) and prolonged (eight doses) ALA treatment of rats (one dose of ALA = 40 mg/kg body weight). The in situ spontaneous chemiluminescence intensity increased 5-fold in brain, 50% in liver and 4-fold in soleus muscle upon two dose-treatment, indicating tissue response to oxidative injury by ALA. Chemiluminescence reached the highest intensity after one or two doses of ALA and decreased after eight doses in all tissues. The plasma trapping capacity, evaluated by the luminol/2-amidinopropane system, gave a parallel response: maximum values after two doses and decreased values after prolonged treatment. After eight doses, the ALA concentration was found to be 3-fold above the normal value in plasma, 48% higher in liver and 38% higher in total brain. These data indicate that the plasma antioxidant system responds to ALA treatment and is correlated with tissue chemiluminescence. In vitro studies showed that ALA does not interfere with the antioxidant plasma capacity, neither promoting oxidation of plasma elements nor binding to plasma proteins.     

Effect of dietary supplementation with a highly pure and concentrated docosahexaenoic acid (DHA) supplement on human sperm function

The aim of this study was to evaluate the possible beneficial effects of diet supplementation with a highly concentrated and purified docosahexaenoic acid (DHA) formula on human sperm function. We performed a prospective, randomized, double blind, placebo-controlled intervention study. One-hundred eighty human semen samples from sixty infertile patients recruited in a private assisted reproduction center were included. All samples were examined according to World Health Organization guidelines. We analyzed macroscopic and microscopic sperm parameters, oxidative stress, apoptosis, lipid peroxidation, mitochondrial membrane potential and DNA fragmentation before and after supplementation with different DHA daily doses (0.5, 1 and 2?g) or placebo for 1 and 3 months. No differences were found in traditional sperm parameters except for progressive sperm motility, with a significant increase after DHA ingestion after the first month with 1 or 2?g doses and after 3 months with 0.5?g of DHA. This effect was more evident in asthenozoospermic patients. No differences were found in any molecular semen parameter except oxidative stress, in which a slight benefit was observed after DHA treatment. In conclusion, this study support previous indications that highlight the importance of DHA supplementation as a means of improving sperm quality in asthenozoospermic men.     

Monosodium glutamate for accidental,retrospective, and medical dosimetry using electron spin resonance

The risk of a radiation episode has increased in the last years due to several reasons. In case of a nuclear incident, as with the use of an improvised nuclear device, determination of the radiation doses received by the victims is of utmost importance to define the appropriate medical treatment or to monitor the late effects of radiation. Dose assessment in case of accidents can be performed using commonplace materials found in the accident area. In this paper, the dosimetric properties of monosodium glutamate are investigated by electron spin resonance spectroscopy (ESR), for retrospective and accidental dosimetry. The spectroscopic parameters were optimized to achieve higher signal intensity and better signal-to-noise ratio. As a result, the lowest detectable dose was 0.1 Gy, and monosodium glutamate showed a linear dose–response curve for doses ranging from 0.1 Gy to 10 kGy. The dosimetric signal was monitored from minutes right after irradiation, until 1 year. No changes in the signal intensity were observed over this period, meaning that doses could be assessed immediately after radiation exposure and can still be reconstructed long after the accident. This property also implies that late effects due to victim’s radiation exposure could be better monitored and understood. ESR signal intensity for samples irradiated with a photon energy below 100 keV was decreased by only 27% and no dose-rate dependence was noticed. Therefore, the ability to measure doses as low as 0.1 Gy, the high stability of the dosimetric signal, as well as independence on dose rate, tissue equivalence, low-cost, and wide commercial availability make monosodium glutamate a very good dosimetric material not only for retrospective and accidental but also for medical dosimetry.     

The radiation crosslinked films based on PLLA/PDLA stereocomplex after TAIC absorption in supercritical carbon dioxide

The absorption of triallyl isocyanurate (TAIC) to equimolar stereo blends of PLLA and PDLA (sb-PLA samples) with crosslinker (TAIC) was controlled by supercritical carbon dioxide (sc-CO₂) treatment. The well-mixed sb-PLA/TAIC (sc-PLA) samples containing certain ratios of TAIC were obtained after vacuum evaporation. The sc-PLA samples were become much softer by sc-CO₂ treatment. The crosslinked sc-PLA materials with different crosslinking density were prepared by irradiation treatment at different radiation doses. Gel fraction, thermal properties and mechanical properties of crosslinked samples were investigated with different sc-PLA samples irradiated at the same radiation dose of 30 kGy and sc-PLA3 irradiated at different radiation doses. The crosslinking network inhibited crystallization, and enhanced the rigidity as well as lowered brittleness of irradiated samples. Although tensile strength and Young’s modulus of sc-PLA are reduced by sc-CO₂ treatment, they are much improved by radiation crosslinking at a suitable dose and the crosslinked samples become harder and tougher. The typical crosslinked obtained from sc-PLA3 irradiated at 30 kGy reveals the best thermal stability with the improved mechanical properties.     

Plasma estriol levels after intramuscular injection of estriol and two of its esters

Twelve female volunteers from Berlin and 9 from Stockholm, all using a contraceptive pill (30 micrograms ethinyl estradiol and 150 micrograms levonorgestrel), received an intramuscular injection of estriol (E3; 1 mg in oil) on day 5 of withdrawal bleeding. Blood samples were collected at increasing time intervals during 4 weeks. Three months later, on day 5 of their withdrawal bleeding, 6 women were given intramuscularly (in oil) estriol 3,17-dipropionate (E3-prop) and 15 women estriol 3,17-dihexanoate (E3-hex). The doses were equivalent to 5 mg of estriol, i.e. 6.94 and 8.90 mg, respectively. Blood samples were collected during a period of 9 weeks. Estriol was analyzed by radioimmunoassay in all plasma samples. The average half-life of E3 ranged from 1.5 to 5.3 h after the administration of E3. It was 12.7 h and between 187 and 221 h after the administration of E3-prop and E3-hex, respectively. The average areas under the curve (in nmol.l-1.h) of E3 were between 82.5 and 161 after the administration of E3-prop or E3-hex, and between 27.1 and 37.9 when E3 had been given. As E3 was administered in a 5-fold lower dose than the esters, the areas under curve appeared to be comparable. Thus, the total exposure to E3 seemed to be almost independent of the type of E3 derivatization, while the time and intensity of exposure were very different.     

Pneumococcal antibody concentrations and carriage of pneumococci more than 3 years after infant immunization with a pneumococcal conjugate vaccine

Background

A 9-valent pneumococcal conjugate vaccine (PCV-9), given in a 3-dose schedule, protected Gambian children against pneumococcal disease and reduced nasopharyngeal carriage of pneumococci of vaccine serotypes. We have studied the effect of a booster or delayed primary dose of 7-valent conjugate vaccine (PCV-7) on antibody and nasopharyngeal carriage of pneumococci 3–4 years after primary vaccination.

Methodology/Principal Findings

We recruited a subsample of children who had received 3 doses of either PCV-9 or placebo (controls) into this follow-up study. Pre- and post- PCV-7 pneumococcal antibody concentrations to the 9 serotypes in PCV-9 and nasopharyngeal carriage of pneumococci were determined before and at intervals up to 18 months post-PCV-7. We enrolled 282 children at a median age of 45 months (range, 38–52 months); 138 had received 3 doses of PCV-9 in infancy and 144 were controls. Before receiving PCV-7, a high proportion of children had antibody concentrations >0.35 µg/mL to most of the serotypes in PCV-9 (average of 75% in the PCV-9 and 66% in the control group respectively). The geometric mean antibody concentrations in the vaccinated group were significantly higher compared to controls for serotypes 6B, 14, and 23F. Antibody concentrations were significantly increased to serotypes in the PCV-7 vaccine both 6–8 weeks and 16–18 months after PCV-7. Antibodies to serotypes 6B, 9V and 23F were higher in the PCV-9 group than in the control group 6–8 weeks after PCV-7, but only the 6B difference was sustained at 16–18 months. There was no significant difference in nasopharyngeal carriage between the two groups.

Conclusions/Significance

Pneumococcal antibody concentrations in Gambian children were high 34–48 months after a 3-dose primary infant vaccination series of PCV-9 for serotypes other than serotypes 1 and 18C, and were significantly higher than in control children for 3 of the 9 serotypes. Antibody concentrations increased after PCV-7 and remained raised for at least 18 months.     

Spontaneous and stimulated interleukin-6 and tumor necrosis factor-alpha production at delivery and three months after birth

OBJECTIVE: To study the production and interrelations of maternal and neonatal cytokines (IL-6 and TNF-alpha) during labor, after vaginal delivery and at three months after delivery. METHOD: The unstimulated concentrations of cytokines in the supernatants of whole-blood cultures and concentrations after PMA (phorbol 12-myristate 13-acetate) and concanavalin (conA) stimulation were determined by enzyme-linked immunosorbent assays (ELISAs). The blood samples were from the peripheral veins of 27 healthy women during term labor and immediately after delivery and three months after delivery. Neonatal samples were taken at birth (cord blood) and three months after delivery. RESULTS: IL-6 responses to stimulation were increased in the parturients and in umbilical cord blood at delivery compared with maternal and neonatal samples obtained 3 months postpartum. In contrast, the production of maternal TNF-alpha in peripheral blood was down-regulated at delivery compared with values 3 months postpartum. After an IL-6 and TNF-alpha burst in umbilical cord samples, neonatal cytokine production was at a low level three months after delivery. IL-6 production tended to be higher in both umbilical cord blood as well as in maternal samples after delivery in women who were younger. In addition, TNF-alpha production in umbilical cord blood was significantly higher in those women who were younger. CONCLUSIONS: The production of IL-6 was up-regulated in both the maternal and in umbilical cord blood at delivery. The production of TNF-alpha was up-regulated in umbilical cord blood compared with neonatal values 3 months after birth. Maternal age had effects on IL-6 and TNF-alpha production at delivery.     

New approach for discrimination of Vibrio vulnificus by real-time PCR before and after gamma-irradiation

The effects of gamma-irradiation on the destruction of Vibrio vulnificus by real-time PCR were studied. gamma-irradiation was found to result in extensive reduction in the molecular size of DNA. Irradiation of viable cells (1 x 10(6) CFU/ml) at 1.08 KGy resulted in 100% destruction determined by plate counts, with most of the DNA from the irradiated cells having a bp-length of less than 1000. The use of a pair of primers to amplify a 1000-bp sequence of DNA from cells exposed to 1.08 KGy failed to yield amplification. In contrast, primers designed to amplify sequences of 700, 300, and 70-bp yielded amplification with C(t) values resulting in 13.4, 27.6, and 45.4% detection of genomic targets. When viable cells of V. vulnificus were exposed to 1.08, 3.0, and 5.0 kGy, the average molecular size of genomic DNA visualized in an agarose gel decreased with increasing dose, corresponding to an increased probability of amplification with primers targeting sequences of decreasing size.     

Improvement of stored turkey semen quality as a result of He-Ne laser irradiation

Two experiments were carried out to evaluate the effects of He-Ne laser irradiation at various energy doses on the quality of stored turkey semen. Four semen pools were used in Experiment 1. Each pool was divided into 10 aliquots, nine of which were irradiated with energy doses ranging from 0.144 to 10.8 J/cm2 while the tenth one was not irradiated (control). Each sample was evaluated for motility immediately after irradiation, 24 and 48 h later. Energy doses ranging from 3.24 to 5.4 J/cm2 had higher (P <0.01) sperm motility index (SMI) value compared to the control and samples irradiated with lower and higher laser doses. The energy dose of 3.96 J/cm2 was selected for Experiment 2 to obtain further insight on its effects on turkey sperm preservation for up to 60 h. Each pool of four semen was divided into two aliquots: one represented the control and the other one was irradiated with He-Ne laser at an energy dose of 3.96 J/cm2. Each sample was evaluated for motility and viability immediately after irradiation and then at 12 h intervals up to 60 h. The cell energy charge was also measured by HPLC. Exposure to 3.96 J/cm2 increased the SMI and viability of turkey semen stored for 60 h compared to the control (P <0.05). The cell energy charge of irradiated samples was 200% higher than in the control. Laser irradiation increased the longevity of stored turkey spermatozoa, and might be a useful technique to enhance semen quality in long-term storage.     

Seroconversion of Hepatitis B Vaccine in Young Children in the Kassena Nankana District of Ghana: A Cross-Sectional Study

Background

Hepatitis B Virus (HBV) infection is an important public health problem that requires high priority efforts towards prevention and control. Active immunization is the single most important and effective preventive measure against HBV infection. As a protective measure, Ghana introduced the mass immunization program against hepatitis B infection in children in 2002 in her Expanded Programme on Immunization (EPI). This study evaluated seroconversion (the point in time when the amount of antibody in the blood becomes detectable) and seroprotection (the point in time when the amount of antibody in the blood is enough to confer protection from the antigen that induced it production) status of children under this mass immunization program and measured their antibody levels five years after immunization.

Materials and Method

200 archived plasma samples of children between the ages of 1–10 years were retrieved from a previous cross-sectional study by researchers from NHRC between 2009 and 2010. Of these, 104 have completed the EPI and were screened for HBsAg. Those found to be HBsAg-seronegative were stratified into three groups according to their age at which the last vaccine was administered. Their anti-HBsAg titer levels were estimated by enzyme linked immunosorbant assay (ELISA).

Results

Two (1.9%) samples were HBsAg seropositive and were excluded from further analyses. 10 more samples were excluded from analyses because they were insufficient. The anti-HBs titers recorded ranged from 1.021 IU/L to 751.64 IU/L indicating a 100% seroconversion rate. In group one (0–6 months), 87.9% were seroprotected. Group two (2-3yrs) had 78.3% seroprotection and group three (3-5yrs) had 41.7% seroprotection. There was no significant difference between group 1 and 2. However, there was a significant difference between group 1 and 3 (p = 0.0137) and between group 2 and 3 (p = 0.0390) respectively. There was no significant difference between male and female children.

Conclusion

All the children who received doses of hepatitis B vaccine at 6, 10 and 14 weeks in the immunization program seroconverted, but their levels of protection waned with increasing years. Booster doses are therefore recommended after 5 years.     

Effects of trichloroethylene,tetrachloroethylene and dichloromethane on enzymatic activities in soil

Summary Enzyme assays for -glucosidase, -acetylglucosaminidase, phosphatase, phosphodiesterase, and proteinase were made in soil samples incubated for two months after contamination with trichloroethylene, tetrachloroethylene, and dichloromethane. These volatile chlorinated hydrocarbons were added at doses of 10, 100, and 1000 g per 100 g dry soil, respectively. Almost no effect was observed in soil sample contaminated with 10 g of the chemicals when compared with control soil. When 100 g of the volatile chlorinated hydrocarbons was added, the activity of -glucosidase, -acetylglucosaminidase and, in part, also of proteinase, was reduced during the first 28 days of incubation but returned to the same or slightly higher level than in the control soil after 2 months. Trichloroethylene, tetrachloroethylene, and dichloromethane at a concentration of 1000 g per 100 g soil primarily inhibited activity of all enzymes under test. However, after two months, the enzymatic activities especially in soil samples contaminated with tetrachloroethylene and dichloromethane were found to be at the same or higher level than in the control soil.     

Application of combined biocides and gamma radiation for keeping good quality stored grapefruits

Grapefruits were irradiated with gamma rays at doses of 0.4, 0.8, 1.6 and 3.2 KGy. Irradiated and non-irradiated fruits were soaked in biocides formulated from essential oils of fennel, peppermint and caraway oil. All treated and non-treated fruits were stored at room temperature (20 ± 1°C) for a period of 70 days. Samples were taken every 14 days for different quality determinations. Non-treated fruits were decayed continuously alongside the different storage periods. However, exposing the studied fruits to gamma radiation induced a promising effect for retarding this decay. Moreover, soaking citrus fruits in the formulated biocides induced further interruption of fruit decay. In addition, the applied treatment exhibited other positive effects for keeping the good quality of the studied fruits during storage.     

Pollution of soil and vegetation in the Thriasian Plain,Greece

Summary The Thriasian Plain near Athens in Greece is a site of concentrated industrial development. Total concentrations of Pb, Cd and extractable SO₄ ^2– in surface soil samples collected from this area, were found to be, respectively, 2–7, 6–34 and 2–20 times higher than those in similar soil samples from parts of the country remote from industrial activity.Total concentrations of Pb, Cd, Zn, Cr and S in samples of olive leaves from the same area were found to be, respectively, 4–40, 3–10, 3–9, 2–6 and 17–21 times higher than those found in samples of olive leaves from rural sites. Leaves of cabbages growing in the area contained between 0.82 and 40 g/g (wet weight) of Pb.Addition of Cd, Ni and Cu to a calcareous potted soil at concentrations of 100, 200 and 200 ppm increased the concentrations of the metals in the needles of Aleppo pine (Pinus halepensis) seedlings to 4.5, 3.5 and 10 ppm, respectively, after 7 months growth in the nursey.