Harvesting and dewatering yeast by microflotation

Microbubble has been applied for the recovery of yeast cells from their growth medium using the bioflocculant–chitosan. Results reaching 99% cell recovery were obtained under various conditions examined. The result of bubble size distribution showed that mean bubble size increased as microbubble diffuser pore size was increased. Also, cell recovery efficiency was a function of both bubble size and particle size (cell size). For smaller particles (<50 μm), relatively smaller bubbles (<80 μm) were found to be more effective for recovery, otherwise, relatively larger bubbles (80–150 μm) proved to be efficient in recovering larger particles (particle size: ∼250 μm). Acidic and neutral pHs were effective in separation as hydrophobic particles were formed. As pH tends toward alkalinity, flocs become more hydrophilic, leading to low recovery from the aqueous solution. In addition, separation efficiency was dependent on flocculant dose as increase in concentration improved flocculation and consequently, yeast recovery. However, above a critical concentration, overdosing occurred and inadvertently, recovery efficiency decreased. The application of chitosan as a bioflocculant and the subsequent application of microflotation for the separation of yeast cells proved effective and promises several advantages over non-bubble based separation techniques that preclude continuous industrial-scale production.     

NH3 gas absorption and bio-oxidation in a single bioscrubber system

A combined ammonia gas absorption and nitrification was conducted in a single bioscrubber. The reactor was consisted of a bubble column (gas absorption) and a packed bed (nitrification) which contained poly-urethane foams with immobilized nitrifying activated sludge. The entering gas and scrubbing liquid were contacted countercurrently. The bubble column elimination capacity (EC) was 26.74 g NH₃/m³ h at >99% ammonia gas removal and effluent gas concentration lower than 2 ppm_v. Without ammonium supplement, EC can reach 35.66 g NH₃/m³ h which is equivalently the highest tolerable ammonia loading rate of 700 g N/m³ day (1650 mg N/L) at the packed bed. At this level, 593 g N/m³-day ammonia removal rate was achieved via nitrification, dominated by ammonia oxidation. Partial recycling (R/Q = 0.5) of scrubbing solution reduced the secondary wastewater volume by producing 233% more concentrated nitrified products. Hydraulic retention time (HRT) of 24 h was found optimal for both processes (gas absorption and nitrification).     

Effect of surface contaminants on oxygen transfer in bubble column reactors

Gas hold-up (ɛ_g), sauter mean bubble diameter (d₃₂) and oxygen transfer coefficient (k_La) were evaluated at four different alkane concentrations (0.05, 0.1, 0.3 and 0.5 vol.%) in water over the range of superficial gas velocity (u_g) of (1.18–23.52) × 10⁻³ m/s at 25 °C in a laboratory-scale bubble column bioreactor. Immiscible hydrocarbons (n-decane, n-tridecane and n-hexadecane) were utilized in the experiments as impurity. A type of anionic surfactant was also employed in order to investigate the effect of addition of surfactant to organic-aqueous systems on sauter mean bubble diameter, gas hold-up and oxygen transfer coefficient. Influence of addition of alkanes on oxygen transfer coefficient and gas hold-up, was shown to be dependent on the superficial gas velocity. At superficial gas velocity below 0.5 × 10⁻³ m/s, addition of alkane in air–water medium has low influence on oxygen transfer coefficient and also gas hold-up, whereas; at higher gas velocities slight addition of alkane increases oxygen transfer coefficient and also gas hold-up. Increase in concentration of alkane resulted in increase in oxygen transfer coefficient and gas hold-up and roughly decrease in sauter mean bubble diameter, which was attributed to an increase in the coalescence-inhibiting tendency in the presence of surface contaminant molecules. Bubbles tend to become smaller with decreasing surface tension of hydrocarbon, thus, oxygen transfer coefficient increases due to increasing of specific gas–liquid interfacial area (a). Empirical correlations were proposed for evaluating gas hold-up as a function of sauter mean bubble diameter, superficial gas velocity and interfacial surface tension as well as evaluating Sherwood number as a function of Schmidt, Reynolds and Bond numbers.     

An alternative explanation for cyanobacterial scum formation and persistence by oxygenic photosynthesis

The cause of persistent cyanobacteria scum formation in lakes is an unresolved subject. Scum refers to the event in which cyanobacteria are at the water surface of a lake. Factors like low turbulence levels, long day-light, high water temperatures and the buoyant capacity of cyanobacterial cells play a role in the occurrence of scums. However, they do not explain why scums are observed at periods during the day when according to theory they should have disappeared into the deeper water layers. In this study, we present an alternative explanation. The hypothesis we present here is that irreversible buoyancy of cyanobacteria colonies is created by the growth of gas bubbles on or within the mucilage of the colonies. These bubbles grow under oxygen super-saturated conditions. At low wind speed and high chlorophyll levels, the dissolved oxygen (DO) produced during photosynthesis by cyanobacteria, cannot escape sufficiently fast to the atmosphere hence a DO supersaturated condition arises in the water. At this stage, growth of oxygen bubbles may occur inside or attached to the mucilage. We present results of compression experiments to support our hypothesis. In a chamber, the pressure on lake water containing a natural cyanobacteria population is increased. At 3 × 10⁵ and 4 × 10⁵ Pa the cyanobacteria colonies were not able to float anymore and sank. This pressure is lower than the 10⁶ Pa needed to collapse all gas vacuoles inside the cyanobacteria cells (Walsby, 1994). The observed change from floating to sinking colonies due to increased water pressure suggests that gas bubbles were present inside the colonies. In lakes, these gas bubbles may lead to permanent buoyancy, i.e. a persistent scum.     

Impact of 1.8-GHz radiofrequency radiation (RFR) on DNA damage and repair induced by doxorubicin in human B-cell lymphoblastoid cells

In the present in vitro study, a comet assay was used to determine whether 1.8-GHz radiofrequency radiation (RFR, SAR of 2 W/kg) can influence DNA repair in human B-cell lymphoblastoid cells exposed to doxorubicin (DOX) at the doses of 0 μg/ml, 0.05 μg/ml, 0.075 μg/ml, 0.10 μg/ml, 0.15 μg/ml and 0.20 μg/ml. The combinative exposures to RFR with DOX were divided into five categories. DNA damage was detected at 0 h, 6 h, 12 h, 18 h and 24 h after exposure to DOX via the comet assay, and the percent of DNA in the tail (% tail DNA) served as the indicator of DNA damage. The results demonstrated that (1) RFR could not directly induce DNA damage of human B-cell lymphoblastoid cells; (2) DOX could significantly induce DNA damage of human B-cell lymphoblastoid cells with the dose–effect relationship, and there were special repair characteristics of DNA damage induced by DOX; (3) E–E–E type (exposure to RFR for 2 h, then simultaneous exposure to RFR and DOX, and exposure to RFR for 6 h, 12 h, 18 h and 24 h after exposure to DOX) combinative exposure could obviously influence DNA repair at 6 h and 12 h after exposure to DOX for four DOX doses (0.075 μg/ml, 0.10 μg/ml, 0.15 μg/ml and 0.20 μg/ml) in human B-cell lymphoblastoid cells.     

Chemical characterization,in vitro dry matter and ruminal crude protein degradability and microbial protein synthesis of some cowpea (Vigna unguiculata L. Walp) haulm varieties

A study was carried out to evaluate the chemical composition, in vitro apparently and truly degraded dry matter (DM), utilizable crude protein at the duodenum (uCP) (total CP at the duodenum minus endogenous CP), methane production, and short chain fatty acid production of haulms of six cowpea varieties. The study was arranged in a 2 × 2 × 2 factorial design, with three replicates. Three improved (ITA2, ITA6 and ITA8) and three commercial (Oloyin, Peu and Sokoto) cowpea varieties harvested during wet and dry seasons were used for the study. After an initial gas test to evaluate 96 h gas production profiles of haulms with and without polyethylene glycol (PEG), the time to half maximal gas production was calculated and a second incubation conducted with fermentation stopped at substrate specific half time (t_1/2) and 24 h for each substrate. True DM degradability was measured from incubated residues and combined with gas volume to estimate the partitioning factor. Crude protein flow to the duodenum was estimated by combining gas volume with the measured ammonia nitrogen in the incubated fluid. Addition of PEG did not have any effect (P>0.05) on all the variables determined. Interaction between group (improved vs. commercial) and season was observed for CP (P=0.002), lignin (P=0.003) and hemicellulose (P=0.030) contents of the haulms. A group × season interaction was also observed for some of the variables at both substrate specific t_1/2 and 24 h. Commercial cowpea haulms had greater (P=0.002) microbial mass and produced less (P<0.05) methane than the improved cowpea haulms. The improved cowpea haulms were less (P<0.001) degraded in the rumen and as a result ensured greater (P<0.001) amount of uCP. The results validated that cowpea haulm is an important agro-based by-product that is adequate in protein and energy to sustain ruminant animal production in Nigeria and other Sub-Saharan African countries during the extended dry season.     

Body mass index,height and early-onset basal cell carcinoma in a case-control study

IntroductionBasal cell carcinoma (BCC) is the most common malignancy in the US. Body mass index (BMI) and height have been associated with a variety of cancer types, yet the evidence regarding BCC is limited. Therefore, we evaluated BMI and height in relation to early-onset BCC (under age 40) and explored the potential role of ultraviolet (UV) radiation exposure and estrogen-related exposures in the BMI-BCC relationship.MethodsBCC cases (n = 377) were identified through a central dermatopathology facility in Connecticut. Control subjects (n = 389) with benign skin conditions were randomly sampled from the same database and frequency matched to cases on age (median = 36, interquartile range 33–39), gender, and biopsy site. Participants reported weight (usual adult and at age 18), adult height, sociodemographic, phenotypic, and medical characteristics, and prior UV exposures. We calculated multivariate odds ratios (ORs) and 95% confidence intervals (CIs) using unconditional logistic regression models.ResultsAdult BMI was inversely associated with early-onset BCC (obese vs. normal OR = 0.43, 95% CI = 0.26–0.71). A similar inverse association was present for BMI at age 18 (OR = 0.54, 95% CI = 0.34–0.85). Excluding UV exposures from the BMI models and including estrogen-related exposures among women only did not alter the association between BMI and BCC, indicating limited mediation or confounding. We did not observe an association between adult height and BCC (OR per cm = 1.00, 95% CI = 0.98–1.02).ConclusionsWe found a significant inverse association between BMI and early-onset BCC, but no association between height and BCC. This association was not explained by UV exposures or estrogen-related exposures in women.     

An efficient synthesis and biological screening of benzofuran and benzo[d]isothiazole derivatives for Mycobacterium tuberculosis DNA GyrB inhibition

A series of twenty eight molecules of ethyl 5-(piperazin-1-yl)benzofuran-2-carboxylate and 3-(piperazin-1-yl)benzo[d]isothiazole were designed by molecular hybridization of thiazole aminopiperidine core and carbamide side chain in eight steps and were screened for their in vitro Mycobacterium smegmatis (MS) GyrB ATPase assay, Mycobacterium tuberculosis (MTB) DNA gyrase super coiling assay, antitubercular activity, cytotoxicity and protein–inhibitor interaction assay through differential scanning fluorimetry. Also the orientation and the ligand–protein interactions of the top hit molecules with MS DNA gyrase B subunit active site were investigated applying extra precision mode (XP) of Glide. Among the compounds studied, 4-(benzo[d]isothiazol-3-yl)-N-(4-chlorophenyl)piperazine-1-carboxamide (26) was found to be the most promising inhibitor with an MS GyrB IC₅₀ of 1.77 ± 0.23 μM, 0.42 ± 0.23 against MTB DNA gyrase, MTB MIC of 3.64 μM, and was not cytotoxic in eukaryotic cells at 100 μM. Moreover the interaction of protein–ligand complex was stable and showed a positive shift of 3.5 °C in differential scanning fluorimetric evaluations.     

Effect of thermal stress on metabolic and oxidative stress biomarkers of Hoplosternum littorale (Teleostei,Callichthyidae)

The present study aimed to investigate in Hoplosternum littorale (Hancock, 1828) the effects of different water temperatures (10 °C, 25 °C-control group- and 33 °C) on physiologic and metabolic traits following acute (1 day) and chronic (21 days) exposures. We analyzed several biomarker responses in order to achieve a comprehensive survey of fish physiology and metabolism under the effect of this natural stressor. We measured morphological indices, biochemical and hematological parameters as well as oxidative stress markers. To evaluate energy consumption, muscle and hepatic total lipid, protein and glycogen concentrations were also quantified. Extreme temperatures exposures clearly resulted in metabolic adjustments, being liver energy reserves and plasma metabolites the most sensitive parameters detecting those changes. We observed reduced hepatosomatic index after acute and chronic exposure to 33 °C while glycogen levels decreased at both temperatures and time of exposure tested. Additionally, acute and chronic exposures to 10 °C increased liver lipid content and plasma triglycerides. Total protein concentration was higher in liver and lower in plasma after chronic exposures to 10 °C and 33 °C. Acute exposition at both temperatures caused significant changes in antioxidant enzymes tested in the different tissues without oxidative damage to lipids. Antioxidant defenses in fish failed to protect them when they were exposed for 21 days to 10 °C, promoting higher lipid peroxidation in liver, kidney and gills. According to multivariate analysis, oxidative stress and metabolic biomarkers clearly differentiated fish exposed chronically to 10 °C. Taken together, these results demonstrated that cold exposure was more stressful for H. littorale than heat stress. However, this species could cope with variations in temperature, allowing physiological processes and biochemical reactions to proceed efficiently at different temperatures and times of exposure. Our study showed the ability of H. littorale to resist a wide range of environmental temperatures and contributes for the understanding of how this species is adapted to environments with highly variable physicochemical conditions.     

Rise and fall of toxic benthic freshwater cyanobacteria (Anabaena spp.) in the Eel river: Buoyancy and dispersal

Benthic cyanobacteria in rivers produce cyanotoxins and affect aquatic food webs, but knowledge of their ecology lags behind planktonic cyanobacteria. The buoyancy of benthic Anabaena spp. mats was studied to understand implications for Anabaena dispersal in the Eel River, California. Field experiments were used to investigate the effects of oxygen bubble production and dissolution on the buoyancy of Anabaena dominated benthic mats in response to light exposure. Samples of Anabaena dominated mats were harvested from the South Fork Eel River and placed in settling columns to measure floating and sinking velocities, or deployed into in situ ambient and low light treatments to measure the effect of light on flotation. Floating and sinking occurred within minutes and were driven by oxygen bubbles produced during photosynthesis, rather than intracellular changes in carbohydrates or gas vesicles. Light experiment results showed that in a natural ambient light regime, mats remained floating for at least 4 days, while in low light mats begin to sink in <24 h. Floating Anabaena samples were collected from five sites in the watershed and found to contain the cyanotoxins anatoxin-a and microcystin, with higher concentrations of anatoxin-a (median 560, max 30,693 ng/g DW) than microcystin (median 30, max 37 ng/g DW). The ability of Anabaena mats to maintain their buoyancy will markedly increase their downstream dispersal distances. Increased buoyancy also allows toxin-containing mats to collect along shorelines, increasing threats to human and animal public health.     

High efficiency production of astaxanthin by autotrophic cultivation of Haematococcus pluvialis in a bubble column photobioreactor

Haematococcus pluvialis was cultivated under photoautotrophic conditions in a bubble column with fed-batch addition of nutrients, especially nitrate, and a cell number above 5 × 10⁶ cells mL⁻¹ was attained after 300 h.The reduction of nutrient concentrations accompanied by dilution of the fermentation broth and an increase in the light intensity enhanced accumulation of astaxanthin. The final astaxanthin concentration of 390 mg L⁻¹ was several times higher than ever reported. This combination of fed-batch addition of nutrients and dilution of broth for nutrient deficiency is a promising method for attainment of high cell and astaxanthin concentrations in a bubble column photobioreactor.     

Temperature and osmotic stress dependence of the thermodynamics for binding linker histone H10, Its carboxyl domain (H10-C) or globular domain (H10-G) to B-DNA

Linker histones (H1) are the basic proteins in higher eukaryotes that are responsible for the final condensation of chromatin. In contrast to the nucleosome core histone proteins, the role of H1 in compacting DNA is not clearly understood. In this study ITC was used to measure the binding constant, enthalpy change, and binding site size for the interactions of H1⁰, or its C-terminal (H1⁰-C) and globular (H1⁰-G) domains to highly polymerized calf-thymus DNA at temperatures from 288 K to 308 K. Heat capacity changes, ΔC_p, for these same H1⁰ binding interactions were estimated from the temperature dependence of the enthalpy changes. The enthalpy changes for binding H1⁰, H1⁰-C, or H1⁰-G to CT-DNA are all endothermic at 298 K, becoming more exothermic as the temperature is increased. The ΔH for binding H1⁰-G to CT-DNA is exothermic at temperatures above approximately 300 K. Osmotic stress experiments indicate that the binding of H1⁰ is accompanied by the release of approximately 35 water molecules.We estimate from our naked DNA titration results that the binding of the H1⁰ to the nucleosome places the H1⁰ protein in close contact with approximately 41 DNA bp. The breakdown is that the H1⁰ carboxyl terminus interacts with 28 bp of linker DNA on one side of the nucleosome, the H1⁰ globular domain binds directly to 7 bp of core DNA, and shields another 6 linker DNA bases, 3 bp on either side of the nucleosome where the linker DNA exits the nucleosome core.     

Bioprocess development of the production of the mutant P-219-L human d-amino acid oxidase for high soluble fraction expression in recombinant Escherichia coli

In order to examine the structure–activity relationship and the substrate specificity of human d-amino acid oxidase (h.DAO), a single amino acid mutation had been established as proline-219-luecine (P-219-L). The gene encoding mutant h.DAO has been cloned and expressed in Escherichia coli BL21 (DE3). It was observed that the host cell was negatively affected by the expressed mutant h.DAO, resulting in a remarkable decrease in the cell growth and consequently the amount of the produced enzyme. To overcome this problem, we investigated several factors that may affect the cell growth rate and the mutant h.DAO production such as optimization of the glucose concentration as a main carbon source and the yeast extract concentration as a main nitrogen source, optimization of dissolved oxygen (DO%) concentration and the addition of benzyl alcohol (BA, which can artificially induce a strong heat shock response at low temperature), to enhance the production of natively folded soluble fraction of the recombinant protein. These parameters were tested on both shake flask level and fed-batch bioreactor level. The Western blot analysis and the enzyme activity assay indicated the higher level of the mutant expression towards enhancement of the conditions by using our designed approach.The specific activity (which was used as an indicator for the level of the desired protein produced = U/mg protein) and the OD_600 nm of the host cells (which was used as an indicator for the cell growth), reached to be 0.061 U/mg protein and 3.44, respectively upon using fed-batch culture system containing the optimized medium composition (15 g/l glucose and 5 g/l yeast extract). While upon using the shake flask level, these values were 0.032 and 1.1, respectively. Enhancement of the cell growth and the enzyme production was noticed after DO% optimization upon using 500 rpm agitation speed and 1.8 v.v.m. (volume volume minute) aeration. The specific activity for the mutant enzyme and the OD_600 nm of the host cells reached to be 0.14 U/mg protein and 7.1, respectively. Finally upon using the optimized culture composition (15 g/l glucose and 5 g/l yeast extract), optimized DO% (using 500 rpm agitation speed and 1.8 v.v.m.) and 0.1 mM BA at the fed-batch bioreactor level, the specific activity and the OD_600 nm of the host cells increased significantly to be 0.21 U/mg protein and 11.3, respectively at 24 h culture. These results indicate the importance of our approaches to overproducing mutant h.DAO in soluble form in E. coli.     

Anti-apoptotic activity of gentiopicroside in d-galactosamine/lipopolysaccharide-induced murine fulminant hepatic failure

This study investigated the hepatoprotective effects of gentiopicroside on d-galactosamine (d-GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were administrated orally with gentiopicroside (40 or 80 mg/kg body weight) at 12 h and 1 h before d-GalN (700 mg/kg)/LPS (10 μg/kg) injection. Gentiopicroside markedly reduced the increases in serum aminotransferase activities and lipid peroxidation. The glutathione content decreased in d-GalN/LPS alone group, and this decrease was attenuated by gentiopicroside. Increases in serum tumor necrosis factor-α (TNF-α), which were observed in d-GalN/LPS alone group, were significantly reduced by gentiopicroside. Importantly, gentiopicroside attenuated d-GalN/LPS-induced apoptosis of hepatocytes, as estimated by the caspase-3 cleavage, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation. d-GalN/LPS-induced caspase-8 and -9 activation was significantly suppressed by gentiopicroside. Moreover, increased cytosolic cytochrome c protein was reduced by gentiopicroside. Also, the increased ratio of Bax and Bcl-2 protein was significantly attenuated by gentiopicroside. After 6 h of d-GalN/LPS injection, phosphorylated c-jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) was significantly increased, whereas phosphorylation JNK and ERK were attenuated by gentiopicroside. Our results suggest that gentiopicroside offers remarkable hepatoprotection against damage induced by d-GalN/LPS related with its anti-apoptotic activities.     

Nitrate removal and greenhouse gas production in a stream-bed denitrifying bioreactor

Denitrifying bioreactors are currently being tested as an option for treating nitrate (NO₃^?) contamination in groundwater and surface waters. However, a possible side effect of this technology is the production of greenhouse gases (GHG) including nitrous oxide (N₂O) and methane (CH₄). This study examines NO₃^? removal and GHG production in a stream-bed denitrifying bioreactor currently operating in Southern Ontario, Canada. The reactor contains organic carbon material (pine woodchips) intended to promote denitrification. Over a 1 year period, monthly averaged removal of influent (stream water) NO₃^? ranged from 18 to 100% (0.3–2.5 mg N L^?1). Concomitantly, reactor dissolved N₂O and CH₄ production, averaged 6.4 μg N L^?1 (2.4 mg N m^?2 d^?1), and 974 μg C L^?1 (297 mg C m^?2 d^?1) respectively, where production is calculated as the difference between inflow and effluent concentrations. Gas bubbles entrapped in sediments overlying the reactor had a composition ranging from 19 to 64% CH₄, 1 to 6% CO₂, and 0.5 to 2 ppmv N₂O; however, gas bubble emission rates were not quantified in this study. Dissolved N₂O production rates from the bioreactor were similar to emission rates reported for some agricultural croplands (e.g. 0.1–15 mg N m^?2 d^?1) and remained less than the highest rates observed in some N-polluted streams and rivers (e.g. 110 mg N m^?2 d^?1, Grand R., ON). Dissolved N₂O production represented only a small fraction (0.6%) of the observed NO₃^? removal over the monitoring period. Dissolved CH₄ production during summer months (up to 1236 mg C m^?2 d^?1), was higher than reported for some rivers and reservoirs (e.g. 6–66 mg C m^?2 d^?1) but remained lower than rates reported for some wastewater treatment facilities (e.g. sewage treatment plants and constructed wetlands, 19,500–38,000 mg C m^?2 d^?1).     

The future excess fraction of occupational cancer among those exposed to carcinogens at work in Australia in 2012

BackgroundStudies in other countries have generally found approximately 4% of current cancers to be attributable to past occupational exposures. This study aimed to estimate the future burden of cancer resulting from current occupational exposures in Australia.MethodsThe future excess fraction method was used to estimate the future burden of occupational cancer (2012–2094) among the proportion of the Australian working population who were exposed to occupational carcinogens in 2012. Calculations were conducted for 19 cancer types and 53 cancer-exposure pairings, assuming historical trends and current patterns continued to 2094.ResultsThe cohort of 14.6 million Australians of working age in 2012 will develop an estimated 4.8 million cancers during their lifetime, of which 68,500 (1.4%) are attributable to occupational exposure in those exposed in 2012. The majority of these will be lung cancers (n = 26,000), leukaemias (n = 8000), and malignant mesotheliomas (n = 7500).ConclusionsA significant proportion of future cancers will result from occupational exposures. This estimate is lower than previous estimates in the literature; however, our estimate is not directly comparable to past estimates of the occupational cancer burden because they describe different quantities – future cancers in currently exposed versus current cancers due to past exposures. The results of this study allow us to determine which current occupational exposures are most important, and where to target exposure prevention.     

Diel activities of antioxidant enzymes,photosynthetic pigments and malondialdehyde content in stationary-phase cells of Tetraselmis gracilis (Prasinophyceae)

The activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as photosynthetic pigment contents and free malondialdehyde (MDA), were determined in senescent batch cultures of Tetraselmis gracilis (Kylin) Butcher, under a cyclic light regime. A 2.6-fold increase in SOD activity (from 53 to 137 U mg⁻¹ protein) was observed in the light phase, contrasting with a 9-fold increase in CAT (from 1 to 9 μmol H₂O₂ min⁻¹ mg⁻¹ protein) and a 1.7-fold increase in APX (from 3 to 5 μmol ascorbate min⁻¹ mg⁻¹ protein) activities, both enzymes peaking in the dark phase. The β-carotene and lycopene content did not vary significantly with the light–dark cycle. The Chl a, Chl b, lutein, zeaxanthin, violaxanthin and neoxanthin pigments exhibited the highest values in the first half (3–6 h) of the light phase, followed by a declining trend and a plateau or a slight increase 3 h from the beginning of the dark phase onwards. The highest values for prasinoxanthin were observed in the second half of the dark phase and the first half of the light phase. None of the pigments displayed any discernible cyclic trend. The possibility of the xanthophyll cycle occurring during senescence is discussed in light of the high value (∼0.9) obtained for the zeaxanthin/(zeaxanthin + violaxanthin) ratio. The free MDA content was enhanced during the experimental period, which may be an indicator of oxidative stress in aging cell cultures. Our results indicated the occurrence of an imbalance between the production of reactive oxygen species and the antioxidant defense in stationary T. gracilis cells.     

Characterization and PCR optimization of the thermostable family B DNA polymerase from Thermococcus guaymasensis

The gene encoding Thermococcus guaymasensis DNA polymerase (Tgu DNA polymerase) was cloned and sequenced. The 2328 bp Tgu DNA polymerase gene encoded a 775 amino acid residue protein. Alignment of the entire amino acid sequence revealed a high degree of sequence homology between Tgu DNA polymerase and other archaeal family B DNA polymerases. The Tgu DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was then purified by heat treatment followed by two steps of chromatography. The optimum pH and temperature were 7.5 and 80 °C, respectively. The optimal buffer for PCR with Tgu DNA polymerase consisted of 50 mM Tris–HCl (pH 8.2), 4 mM MgCl₂, 50 mM KCl, and 0.02% Triton X-100. Tgu DNA polymerase revealed 4-fold higher fidelity (3.17 × 10^?6) than Taq DNA polymerase (12.13 × 10^?6) and a faster amplification rate than Taq and Pfu DNA polymerases. Tgu DNA polymerase had an extension rate of 30 bases/s and a processivity of 150 nucleotides (nt). Thus, Tgu DNA polymerase has some faster elongation rate and a higher processivity than Pfu DNA polymerase. Use of different ratios of Taq and Tgu DNA polymerases determined that a ratio of 4:1 efficiently facilitated long PCR (approximately 15 kb) and a 3-fold lower error rate (4.44 × 10^?6) than Taq DNA polymerase.     

DNA damage induction and antiproliferative activity of vanadium(V) oxido monoperoxido complex containing two bidentate heteroligands

Several peroxidovanadium(V) complexes have been shown as a potent anticancer agents. The aim of this study was to investigate the interaction of monoperoxidovanadium(V) complex Pr₄N[VO(O₂)(ox)(phen)], (Vphen), [phen = 1,10-phenantroline, ox = oxalate(2?) and Pr₄N = tetra(n-propyl)ammonium(1+)] with DNA. UV–Vis spectrophotometry and the alkaline single-cell gel electrophoresis (SCGE, the comet assay) were used to examine the possibility of the vanadium(V) complex to induce changes in DNA. The interaction of Vphen with calf thymus DNA resulted in absorption hyperchromicity in DNA spectrum and shift of the absorption band of DNA to longer wavelengths for the [complex]/[DNA] concentration ratio equals to 4 and after 60 min of incubation. The rise in DNA absorption (by 34%) and bathochromic shift (Δλ_max = 6 nm) are indicative of the interaction between DNA and the complex molecules. DNA strand breaks in cellular DNA were investigated using the comet assay. The human lymphocytes were exposed to various concentrations of Vphen for 30 min. The results revealed that Vphen contributed to the DNA damage expressed as DNA strand breaks in concentration dependent manner. The used concentrations of Vphen (ranging from 0.1 to 100 μmol/L) caused higher DNA damage in lymphocytes compared to untreated cells (from 1.2 times for 0.1 μmol/L to 1.8 times for 100 μmol/L). Vphen was screened for its potential antitumor activity towards murine leukemia cell line L1210. Vphen exhibited significant antiproliferative activity depending on its concentration and time of exposure. The IC₅₀ values were 0.247 μg/mL (0.45 μmol/L) for 24 h, 0.671 μg/mL (1.21 μmol/L) for 48 h and 0.627 μg/mL (1.13 μmol/L) for 72 h.     

Time-lapse videography of human oocytes following intracytoplasmic sperm injection: Events up to the first cleavage division

A total of 341 fertilized and 37 unfertilized oocytes from 63 intracytoplasmic sperm injection (ICSI) treatment cycles were included for retrospective assessment using the Embryoscope™ time-lapse video system. The second polar body (pb2) extrusion occurred at 2.9 ± 0.1 h (range 0.70–10.15 h) relative to sperm injection. All oocytes reduced in size following sperm injection (p < 0.05) with shrinkage ceasing after 2 h in the unfertilized and at pb2 extrusion in the fertilized oocytes. Pb2 extrusion was significantly delayed for women aged >38 years compared to those <35 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01) or 35–38 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01), but timing was not related to the Day 3 morphological grades (1–4) of subsequent embryos (2.9 ± 0.1, 2.9 ± 0.1, 2.8 ± 0.2 and 3.0 ± 0.1; p > 0.05 respectively). A shorter time of first cleavage division relative to either sperm injection or pb2 extrusion is associated with both top grade (AUC = 0.596 or 0.601, p = 0.006 or 0.004) and usable embryos (AUC = 0.638 or 0.632, p = 0.000 respectively) on Day 3. In summary, (i) pb2 of human oocytes extrudes at various times following sperm injection, (ii) the timing of pb2 extrusion is significantly delayed when female age >38 years, but not related to subsequent embryo development, (iii) all human oocytes reduce in size following sperm injection, (iv) completion of pb2 extrusion in the fertilized oocytes is a pivotal event in terminating shrinkage of the vitellus, and (v) time to first cleavage division either from sperm injection or pb2 extrusion is a significant predictive marker for embryo quality on Day 3.