Effect of yeast extract on fluoranthene degradation and aromatic ring dioxygenase expressing bacterial community structure of a fluoranthene degrading bacterial consortium

Fluoranthene (Fla) is a high molecular weight polycyclic aromatic hydrocarbon that exerts hazardous effects on living organisms. An efficient Fla degrading bacterial consortium LP was enriched from an oil contaminated soil sample, with and without yeast extract as a supplement. Objective of the present study was to see if there was any differential effect of yeast extract addition on Fla degradation potential and aromatic ring dioxygenase expressing bacteria (ARDB) of the enrichments. Primary enrichment of the soil sample was carried out in minimal salt medium (MSM) added with 500 mg l⁻¹ Fla and 0.05% yeast extract (YMSM). Secondary, tertiary and subsequent enrichments were prepared in YMSM and MSM after every sixteen days of incubation. Fla was efficiently degraded by YMSM enriched culture than MSM enriched culture. However, when MSM enrichment was incubated longer instead of further subculturings, it also degraded Fla efficiently. All three enrichments exhibited growth of bacterial colonies on Fla sprayed minimal agar plates however only YMSM enrichment showed clear zone forming bacterial colonies. A positive effect was observed of yeast extract on ARDB population of LP consortium. To our limited knowledge this is first time that effect of yeast extract on ARDB population was studied.     

The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

Optimization of the cryopreservation of biological resources,Toxoplasma gondii tachyzoites,using flow cytometry

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10⁶ tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me₂SO without incubation. A cooling rate of ∼1 °C/min to −80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.     

Shifts in thermoregulatory strategy during ontogeny in harp seals (Pagophilus groenlandicus)

Heat balance can be difficult for young and/or small animals in polar regions because environmental conditions in combination with small body size or physiological immaturity can increase heat loss. We investigated how thermoregulatory patterns change with ontogeny in 5 age classes of harp seal (Pagophilus groenlandicus) from birth to post-molt to further understand the timing of thermoregulatory development in relation to their potential vulnerability to ongoing fluctuations in the extent and stability of Arctic pack ice. We measured changes in the amount, conductivity, and resistance of the seal pups׳ insulative layers (blubber and fur), the potential for endogenous heat-generation by shivering (muscle enzyme activity), and nonshivering thermogenesis (NST; brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression and mitochondrial density). There was no significant difference in blubber conductivity among age classes, though the amount of blubber insulation significantly increased from birth to weaning. Pelage conductivity was low (0.12±0.01 W m⁻¹ °C⁻¹) except in 9-day old pups (0.40±0.08 W m⁻¹ °C⁻¹); the significantly higher conductivity may signal the beginning of the molt, and this age group may be the most vulnerable to early water entry. Citrate synthase activity significantly increased (49.68±3.26 to 75.08±3.52 μmol min⁻¹ g wet weight⁻¹) in the muscle; however it is unlikely that increasing a single enzyme greatly impacts heat generation. BAT of younger pups contained UCP1, though expression and mitochondrial density quickly declined, and the ability of pups to produce heat via NST was lost by weaning. While total thermal resistance did not differ, neonatal and early nursing animals gained the majority of their thermal resistance from lanugo (82.5±0.03%); however, lanugo is not insulative when wet, and NST may be important to maintain euthermia and dry the coat if early immersion in water occurs. By late nursing, blubber seems sufficient as insulation (75.87±0.01% of resistance after 4 weeks), but high conductivity of fur may be responsible for retention of UCP1 expression. Weaned animals rely on blubber insulation, and no longer need NST, as wetted fur is no longer a threat to euthermia.     

Sensitivity of human embryonic stem cells to different conditions during cryopreservation

Low cell recovery rate of human embryonic stem cells (hESCs) resulting from cryopreservation damages leads to the difficulty in their successful commercialization of clinical applications. Hence in this study, sensitivity of human embryonic stem cells (hESCs) to different cooling rates, ice seeding and cryoprotective agent (CPA) types was compared and cell viability and recovery after cryopreservation under different cooling conditions were assessed. Both extracellular and intracellular ice formation were observed. Reactive oxidative species (ROS) accumulation of hESCs was determined. Cryopreservation of hESCs at 1 °C/min with the ice seeding and at the theoretically predicted optimal cooling rate (TPOCR) led to lower level of intracellular ROS, and prevented irregular and big ice clump formation compared with cryopreservation at 1 °C/min. This strategy further resulted in a significant increase in the hESC recovery when glycerol and 1,2-propanediol were used as the CPAs, but no increase for Me₂SO. hESCs after cryopreservation under all the tested conditions still maintained their pluripotency. Our results provide guidance for improving the hESC cryopreservation recovery through the combination of CPA type, cooling rate and ice seeding.     

The oxidant-antioxidant equilibrium,activities of selected lysosomal enzymes and activity of acute phase protein in peripheral blood of 18-year-old football players after aerobic cycle ergometer test combined with ice-water immersion or recovery at room temperature

The goal of the study was to evaluate the effect of an aerobic exercise bout followed by ice-water immersion or recovery at room temperature on the redox state, activities of selected lysosomal enzymes and activity of α1-antitrypsin (AAT) in the blood of healthy sportsmen. Eleven amateur football players aged 18 were randomly assigned to two similar 30-min aerobic cycle ergometer tests followed by a recovery at room temperature (20 °C; Experiment 1) or ice-water immersion (3 °C, 5 min; Experiment 2). Peripheral blood was collected three times during both study experiments: before (baseline), as well as 20 and 40 min after the recovery or immersion. The concentrations of thiobarbituric acid reactive substances in blood plasma (plTBARS) and erythrocytes (erTBARS) were measured. The erythrocytic activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were also determined. In the blood serum, the activities of acid phosphatase (AcP), arylsulphatase (ASA), cathepsin D (CTS D) and AAT were evaluated. The activities of AcP, ASA, CTS D and AAT changed similarly during both experiments. The GPx activity decreased 40 min after the exercise/recovery compared to the baseline activity and was lower than 40 min after the exercise/immersion. The exercise followed by the recovery or immersion had no significant effect on the serum lysosomal and AAT activities in the studied men. The exercise/recovery reduced the hydrogen peroxide concentration in the men's erythrocytes, however the exercise/immersion demonstrated the opposite effect.     

Effect of molybdenum on secondary metabolic process of glycyrrhizic acid in Glycyrrhiza uralensis Fisch.

A pot experiment was conducted to investigate into effects of molybdenum (Mo) on the secondary metabolic process of glycyrrhizic acid (GA). One-year-old seedlings were grown in pots with washed vermiculite and sand. Hoagland nutrition solution was irrigated with four concentrations: 0, 0.52, 5.2 and 10.4 mg L⁻¹. The accumulations of GA and its biosynthetic precursors (β-amyrin and squalene) and then expression of the key synthase (β-amyrin synthase, β-AS) were studied on 35, 70 and 105 d. In the early stage, that was on the 35 and 70 d, the contents of squalene and GA, and the expression of β-AS gene under 0.52 and 5.2 mg L⁻¹ Mo treatments were significantly higher than that under 0 and 10.4 mg L⁻¹ Mo. There was a contrary result of β-amyrin. However, the content of squalene under 0 mg L⁻¹ Mo was the highest on 105 d. Thus, it suggested an appropriate concentration of Mo could promote the accumulation of GA, by affecting the biosynthetic process of GA at a certain time. Practically, the time and amount of application of Mo on Glycyrrhiza uralensis should be the noted.     

Decomposition of sugarcane bagasse with lignocellulose-derived thermotolerant and thermoresistant Penicillia and Aspergilli

To promote the decomposition of sugarcane bagasse (SCB) for conversion into value-added products and to reduce waste, the capability of fungal mixes (FMs) to degrade SCB was examined. A total of 169 isolates from SCB and non-SCB were categorized as thermotolerant and thermoresistant. Thirty-six fungal candidates were screened for the presence of polyphenol oxidase, endoglucanase (EDN) and xylanase (XLN) activities, and EDN and XLN activities were quantitated. Five identified isolates (Aspergillus flavus AG10; Aspergillus niger AG68 & NB23; and Penicillium citrinum AG93 & AG140) were selected as the best enzyme producers, and 15 moderately to highly xylolytic, cellulolytic and ligninolytic isolates were added to construct FMs. Using a Taguchi design, the top ten reducing sugar-producing FMs (no. 12 showed the maximum amount of reducing sugar, at 2.11 mg g⁻¹, followed by no. 7, 15, 2, 16, 11, 13, 6, 4, & 8) were selected as potential agents for decomposition durations of 1, 2 and 3 months. The maximum decrease in SCB materials compared with the control was generated by FM 6 (9.08% cellulose reduction); FM 13 (21.03% hemicellulose reduction); and FM 16 (9.21% lignin reduction). These results indicate the potential use of SCB as a substrate for synergistic FMs. These FMs could be applied to the large-scale composting of SCB and other related agricultural residues, thus improving the biological pretreatment of lignocellulose.     

Bioaugmentation of a methyl parathion contaminated soil with Pseudomonas sp. strain WBC-3

Pseudomonas sp. strain WBC-3 utilizes methyl parathion (MP) and para-nitrophenol as the sole source of carbon, nitrogen and energy. In this study, strain WBC-3 was inoculated into lab-scale MP-contaminated soil for bioaugmentation. Accelerated removal of MP was achieved in bioaugmentation treatment compared to non-bioaugmentation treatment, with complete removal of 0.536 mg g⁻¹ dry soil in bioaugmentation treatment within 15 days and without accumulation of toxic intermediates. The analysis of denaturing gradient gel electrophoresis and real-time PCR showed that strain WBC-3 existed stably during the entire bioaugmentation period. Simultaneously, redundancy analysis for evaluating the relationships between the environmental factors and microbial community structure indicated that the indigenous bacterial community structure was significantly influenced by strain WBC-3 inoculation (P = 0.002).     

Effect of resveratrol on vitrified in vitro produced bovine embryos: Recovering the initial quality

Although vitrification is the current routine method for human embryo cryopreservation, it may cause detrimental effects. The aim of this study was to evaluate the effect of supplementing in vitro culture (IVC) media and/or vitrification solutions (VS) with Resveratrol on the presence of apoptotic markers, reactive oxygen species (ROS) level, glutathione (GSH) content and relative gene abundance. Abattoir-derived oocytes were matured and fertilized in vitro according to a standard procedure. Zygotes were cultured in IVC medium supplemented with or without 0.5 μM Resveratrol (C^R, C⁻ respectively). On day 7, blastocysts were vitrified using the minimum volume vitrification method supplementing VS with (C⁻V^R, C^RV^R) or without (C^–V^-, C^RV⁻) 0.5 μM Resveratrol. After warming, embryonic quality parameters were evaluated. Survival rates were significantly lower in C^RV^R group compared with C^RV⁻ group, but no differences in hatching rate were observed between groups. Vitrification/warming process did not alter total cell number or the presence of apoptotic or dead cells, but C^RV⁻ and C^RV^R groups presented a significant increase in dead cells (P < 0.05 by ANOVA). Resveratrol supplementation in VS (C⁻V^R) restored GSH content (P < 0.05) to the level found in the C^R group. Vitrification/warming process significantly increased the expression of FOXO3A, PNPLA2, BCL2L1 and BAX genes (P < 0.05). Resveratrol addition to IVC medium or VS partially compensated this increase for FOXO3A and PNPLA2 (P < 0.05) but not for BCL2L1 and BAX. In conclusion, supplementation of IVC media or VS with 0.5 μM resveratrol may help embryos to partially restore the initial quality they had before the cryopreservation process.     

Effects of different sucrose concentrations on vitrified porcine preantral follicles: Qualitative and quantitative analysis

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 μl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M–0.25 M – 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and a significant increase in the cytoplasmic area of primordial follicles with 0 M sucrose (p < 0.05). The qualitative and the quantitative analysis appear to be a complementary tool when choosing a vitrification protocol. For our cryopreservation system - vitrification of ovarian cortex slices in cryotubes-the best vitrification medium was TCM 199-Hepes with 30% de ethylene glycol, 20% of Fetal Bovine Serum and 0 or 0.25 M sucrose. The present study shows that the use of high sucrose concentrations in the vitrification solution has a deleterious effect on the preservation of porcine preantral follicles contained in ovarian tissue. Consequently, its use at 0.75 M or 1 M wouldn't be recommended.     

Essential oil yield,composition, bioactivity and leaf morphology of Juniperus oxycedrus L. from Bulgaria and Serbia

Juniperus oxycedrus L. (Cupressaceae Bartlett) is widely distributed in countries with a Mediterranean climate. All plant parts contain highly aromatic essential oil (EO) and recently there have been efforts to introduce it as a cultivated crop. The species is known for its large morphological and chemical variation and its debatable taxonomic status. This study aimed to (1) compare content, composition, and antimicrobial activity of J. oxycedrus EO samples from plants growing in Bulgaria and Serbia, and (2) quantify morphological variations of leaves. Тhe EO content (yield) in dried juniper leaves varied from 0.06% (Кopaonik, Serbia) to 0.24% (Markovo, Bulgaria). We identified 51 EO constituents, belonging to monoterpenes, sesquiterpenes, and diterpenes. The class monoterpenes (monoterpene hydrocarbons and oxygenated monoterpenes) were the predominant compounds, representing 38.6–65.4% of the total EO, consisting primarily of α-pinene, limonene, sabinene, β-pinene, and β-myrcene. In addition, α-pinene was the major oil constituent in plants from all locations. Sesquiterpenes (sesquiterpene hydrocarbons and oxygenated sesquiterpenes) were the second largest class of constituents, which represented 19.3% tо 33.6% of the total EO. γ-Elemene was found only in the EO of J. oxycedrus from Bulgaria, while a high concentration of α-curcumene was found only in samples from Serbia (7.5–7.8%). Significant differences in antimicrobial activity of the EO were found in bacterial strains Bacillus cereus and Streptococcus pneumoniae. There was no significant difference among the mean leaf width of the six combinations location x sex, and the overall leaf mean width was 1.24 mm. However, there was a significant difference between the mean leaf lengths. In this study, none of the studied populations had a higher concentration of limonene than of α-pinene, indicating that the flora of the two countries include J. oxycedrus and not the previously reported J. deltoides. The results revealed significant variation in EO profile that may contribute to the development of new cultivars of J. oxycedrus.     

Rhizoremediation of phenol and chromium by the synergistic combination of a native bacterial strain and Brassica napus hairy roots

A bacterial strain resistant to phenol and Cr (VI) was isolated from an industrial polluted soil of Córdoba province (Argentina), which was identified as Pantoea sp. FC 1. This microorganism was able to use phenol as sole carbon source. In addition it was capable of reducing Cr (VI) to Cr (III) in mineral and nutrient media. The isolated strain exhibited some properties as plant-growth promoting bacterium (PGPB), such as production of Indole Acetic Acid (IAA) and synthesis of siderophores, as well as being capable of solubilizing inorganic phosphates. A rhizoremediation system using the association Pantoea sp. FC 1-Brassica napus hairy roots (HRs) was tested for phenol and Cr (VI) removal in a hydroponic system. Microbial inoculation improved both phenol removal and chromium accumulation efficiency by HRs, showing a significant increase in Cr (III) accumulation compared to non-inoculated HRs, exceeding 1000 mg kg⁻¹. Cr (III) was detected in HR biomass and supernatants, suggesting a possible Cr (VI) reducing activity of B. napus HRs. Basic studies in plant model systems, such as HRs, provide additional useful information that could facilitate the transition of this technology into plants suitable for practical rhizoremediation applications.     

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90–180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P < 0.01) and increase the number of IRS2 positive cells and Ob-R positive cells in hippocampus (P < 0.01). ② The expressions of mitochondrial energy-relative proteins, VDAC1 and cytC, were higher in cold-exposure group than in control group with both immunohistochemical staining and Western blot (P < 0.01). ③ Chronic cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P < 0.01). In conclusion, chronic cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance.