Experimental study of microorganism disruption using shear stress

There has been a broad spectrum of theoretical and experimental works on microorganism disruption methods undertaken in the past. However, there is a lack of understanding regarding the actual reasons for microorganism disruption using ultrasound and whether it is caused by shock or shear. In the case of shear stress, which is the focus of this paper, analysis of the intense turbulent flow region of an in-house built shear apparatus combined with the experimental results demonstrated that when the energy dissipation rate in the turbulence region is high, and the size of the eddy is smaller than the size of the cell, the likelihood of yeast disruption is high. The mechanical properties of yeast cells combined with the calculated energy dissipation rate were used to evaluate the yeast disruption efficiency (log reduction). The results show that the shear apparatus can efficiently and effectively disrupt S. cerevisiae at different treatment times, suspension temperatures and rotor speeds. The experimental work suggests that maximum yeast log reduction was achieved when the maximum power dissipation of 2.095 kW was recorded at 10,000 RPM, while suspension temperature was controlled below 35 °C. The corresponding shear stress at 10,000 RPM was 2586.2 Pa.     

2,3 Butanediol production in an obligate photoautotrophic cyanobacterium in dark conditions via diverse sugar consumption

Cyanobacteria are under investigation as a means to utilize light energy to directly recycle CO₂ into chemical compounds currently derived from petroleum. Any large-scale photosynthetic production scheme must rely on natural sunlight for energy, thereby limiting production time to only lighted hours during the day. Here, an obligate photoautotrophic cyanobacterium was engineered for enhanced production of 2,3-butanediol (23BD) in continuous light, 12 h:12 h light-dark diurnal, and continuous dark conditions via supplementation with glucose or xylose. This study achieved 23BD production under diurnal conditions comparable to production under continuous light conditions. The maximum 23BD titer was 3.0 g L⁻¹ in 10 d. Also achieving chemical production under dark conditions, this work enhances the feasibility of using cyanobacteria as industrial chemical-producing microbes.     

A novel rapid method for simultaneous determination of eight active compounds in silymarin using a reversed-phase UPLC-UV detector

A novel rapid chromatographic method based on utilization of UPLC column was developed for the analysis of eight active compounds in silymarin. The analysis was performed on a Waters Acquity UPLC system with an Acquity UPLC^BEH C18 column (5 mm × 2.1 mm I.D., 1.7 μm) and a gradient elution of methanol and water containing 0.01% formic acid with a run time of 9 min, in which the retention time of the last analyte was 5.8 min. And all eight active compounds achieved complete separation. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity. The results indicated that the type of column, the type of mobile phase and the modified addition were significant to the separation of isomeric compounds in herb extracts.     

Thermoimaging as a tool for studying light-induced heating of leaves: Correlation of heat dissipation with the efficiency of photosystem II photochemistry and non-photochemical quenching

Thermoimaging – a highly sensitive and non-invasive method of temperature measurement – was applied to explore the role of changing photosynthetic efficiency in light-induced heating of tobacco (Nicotiana tabacum cv. Samsun) leaves. In the absence of evaporative cooling through the stomata, which was achieved by covering leaves with Vaseline, illumination with 50–1400 μM photons m^?2 s^?1 intensity of photosynthetically active radiation resulted in ≈1–5 °C leaf temperature increase in about 2 min. The heating effect showed a non-linear correlation with the extent of non-photochemical quenching (NPQ) resulting in higher leaf temperatures at higher NPQ values. When leaves were adapted to excessive irradiance (1300 μM photons m^?2 s^?1 for 6 h), which resulted in reduction of photosynthetic efficiency and amplification of NPQ the light-induced heating effect was enhanced. The experimental results have been explained on the basis of a simple theoretical model characterizing the balance of energy fluxes in leaves in relation to the efficiency of photosystem II photochemistry and non-photochemical quenching. The role of alternative energy dissipation pathways outside of PSII in the phenomenon of light-induced leaf heating is also discussed.     

DNA binding acridine–thiazolidinone agents affecting intracellular glutathione

Three new acridine–thiazolidinone derivatives (2a–2c) have been synthesized and their interactions with calf thymus DNA and a number of cell lines (leukemic cells HL-60 and L1210 and human epithelial ovarian cancer cell lines A2780) were studied. The compounds 2a–2c possessed high affinity to calf thymus DNA and their binding constants determined by spectrofluorimetry were in the range of 1.37 × 10⁶–5.89 × 10⁶ M^?1. All of the tested derivatives displayed strong cytotoxic activity in vitro, the highest activity in cytotoxic tests was found for 2c with IC₅₀ = 1.3 ± 0.2 μM (HL-60), 3.1 ± 0.4 μM (L1210), and 7.7 ± 0.5 μM (A2780) after 72 h incubation. The cancer cells accumulated acridine derivatives very fast and the changes of the glutathione level were confirmed. The compounds inhibited proliferation of the cells and induced an arrest of the cell cycle and cell death. Their influence upon cells was associated with their reactivity towards thiols and DNA binding activity.     

Thymoquinone causes multiple effects,including cell death,on dividing plant cells

Thymoquinone (TQ) is a major constituent of Nigella sativa oil with reported anti-oxidative activity and anti-inflammatory activity in animal cells. It also inhibits proliferation and induces programmed cell death (apoptosis) in human skin cancer cells. The present study sought to detect the influence of TQ on dividing cells of three plant systems and on expression of Bcl2-associated athanogene-like (BAG-like) genes that might be involved during the process of cell death. BAG genes are known for the regulation of diverse physiological processes in animals, including apoptosis, tumorigenesis, stress responses, and cell division. Synthetic TQ at 0.1 mg/mL greatly reduced wheat seed germination rate, whereas 0.2 mg/mL completely inhibited germination. An Evans blue assay revealed moderate cell death in the meristematic zone of Glycine max roots after 1 h of TQ treatment (0.2 mg/mL), with severe cell death occurring in this zone after 2 h of treatment. Light microscopy of TQ-treated (0.2 mg/mL) onion hairy root tips for 1 h revealed anti-mitotic activity and also cell death-associated changes, including nuclear membrane disruption and nuclear fragmentation. Transmission electron microscopy of TQ-treated cells (0.2 mg/mL) for 1 h revealed shrinkage of the plasma membrane, leakage of cell lysate, degradation of cell walls, enlargement of vacuoles and condensation of nuclei. Expression of one BAG-like gene, previously associated with cell death, was induced 20 min after TQ treatment in Glycine max root tip cells. Thus, TQ has multiple effects, including cell death, on dividing plant cells and plants may serve as a useful system to further investigate the mechanisms underlying the response of eukaryotic cells to TQ.     

A phycocyanin·phellandrene synthase fusion enhances recombinant protein expression and β-phellandrene (monoterpene) hydrocarbons production in Synechocystis (cyanobacteria)

Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow K_cat (low V_max) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-phellandrene synthesis, yielding an average of 3.2 mg product g⁻¹ dry cell weight (dcw) versus the 0.03 mg g⁻¹ dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-phellandrene hydrocarbons production in these photosynthetic microorganisms.     

Seasonal variations in photosynthetic irradiance response curves of macrophytes from a Mediterranean coastal lagoon

The main photosynthesis and respiration parameters (dark respiration rate, light saturated production rate, saturation irradiance, photosynthetic efficiency) were measured on a total of 23 macrophytes of the Thau lagoon (2 Phanerogams, 5 Chlorophyceae, 10 Rhodophyceae and 6 Phaeophyceae). Those measurements were performed in vitro under controlled conditions, close to the natural ones, and at several seasons. Concomitantly, measurements of pigment concentrations, carbon, phosphorous and nitrogen contents in tissues were performed. Seasonal intra-specific variability of photosynthetic parameters was found very high, enlightening an important acclimatation capacity. The highest photosynthetic capacities were found for Chlorophyceae (e.g. Monostroma obscurum thalli at 17 °C, 982 μmol O₂ g⁻¹ dw h⁻¹ and 9.1 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹, respectively for light saturated net production rate and photosynthetic efficiency) and Phanerogams (e.g. Nanozostera noltii leaves at 25 °C, 583 μmol O₂ g⁻¹ dw h⁻¹ and 2.6 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹ respectively for light saturated net production rate and photosynthetic efficiency). As expected, species with a high surface/volume ratio were found to be more productive than coarsely branched thalli and thick blades shaped species. Contrary to R_d (ranging 6.7–794 μmol O₂ g⁻¹ dw h⁻¹, respectively for Rytiphlaea tinctoria at 7 °C and for Dasya sessilis at 25 °C) for which a positive relationship with water temperature was found whatever the species studied, the evolution of P/I curves with temperature exhibited different responses amongst the species. The results allowed to show summer nitrogen limitation for some species (Gracilaria bursa-pastoris and Ulva spp.) and to propose temperature preferences based on the photosynthetic parameters for some others (N. noltii, Zostera marina, Chaetomorpha linum).     

Partial permeabilisation and depolarization of Salmonella enterica Typhimurium cells after treatment with pulsed electric fields and high pressure carbon dioxide

The aim of the present study is to investigate the efficiency of the combined pulsed electric fields and high pressure carbon dioxide (PEF + HPCD) treatment on the Gram-negative pathogen Salmonella Typhimurium in a liquid medium, by means of both plate count technique and flow cytometry (FCM). PEF was applied at two conditions: (1) 1 single pulse of 1 ms length at 30 kV/cm and (2) 12 pulses of 4 ms length at 30 kV/cm, while HPCD at 12 MPa, 22 °C and 35 °C for different treating times (0–45 min). At both temperatures, the application of PEF as HPCD pre-treatment was demonstrated to enhance the inactivation kinetics and to decrease the treatment time to inactivate S. Typhimurium if compared to HPCD alone. Further, the approach based on FCM permitted to investigate the functional status of bacterial cells after PEF and HPCD treatments distinguishing among viable bacteria (considered as intact cells), permeabilised cells and depolarised cells simultaneously. It has been demonstrated that the synergistic effect is due to the electroporation effect of PEF which lead to changes in the cell membrane potential but also in a partial structural damage, favoring the subsequent CO₂ penetration into the cells and increasing the inactivation kinetics, thus improving the efficiency of the entire process.     

Effects of aqueous ozone on Pseudomonas syringae viability and ice nucleating activity

This study reports the impact of different ozone treatments on a Pseudomonas syringae strain known for its ice nucleation activity (INA). Ozone is a very powerful germicidal agent used for water treatment. The effect of ozone on viability and on cultivability of P. syringae was determined by flow cytometry analysis and by plate counting respectively. The impact of ozone on the outer membrane using the INA as marker was investigated by the drop freezing technique.The destruction curve followed a shoulder pattern with a slight reduction in population with CT values between 0 and 8 min. For an initial population of 9.3 log CFU mL^?1, the cultivability was lost starting at 14 min and a loss of viability was observed after 16 min of ozone treatment at 0.45 mg L^?1. Microscopic observations at this point revealed whole but aggregated bacilli. INA decreased after 8 min of ozone treatment but did not disappear. This decrease could be due to the progressive disruption of ice nucleating sites in the outer membrane. It was however partially restored after long storage at 4 °C of dead cells treated for 16 min.     

A highly efficient hydrophobic interaction chromatographic absorbent improved the purification of hepatitis B surface antigen (HBsAg) derived from Hansenula polymorpha cell

To improve the purification efficiency of recombinant hepatitis B surface antigen derived from Hansenula polymorpha (Hans-HBsAg), a serial of absorbents for hydrophobic interaction chromatography with the controllable ligand density and spacer arm were synthesized, then developed and further applied to purify Hans-HBsAg. The absorbent, Butyl-S QZT with the ligand density of 25 μmol/(g wet gel) and spacer arm of 3C, was screened out and its physical and chemical properties were evaluated. High rigidity and low backpressure (<0.06 MPa) were obtained at the flow rate up to 20 ml/min. Moreover, it has the stable chemical characteristics of subjecting to high concentrations of acid, alkali and detergents. This HIC absorbent was further applied to purify Hans-HBsAg with the recovery 94% and purification-fold 9 under the optimized operation condition at pH 6.5 and concentration of ammonium sulfate 7.5%. Finally, the HIC adsorbent of Butyl-S QZT was applied in the integrated three-step chromatographic purification process to purify Hans-HBsAg. About 140 mg of purified Hans-HBsAg was obtained from 1 l cell disruption supernatant at the total recovery of 27% and the purification-fold of 151.8. Based on the assay of SDS-PAGE and SEC-HPLC, the purity of the purified HBsAg was over 99% to meet the requirement for the further inoculation use.     

New HPLC–MS method for the simultaneous quantification of the antileukemia drugs imatinib,dasatinib, and nilotinib in human plasma

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 μl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia.     

Discovery of novel 4-anilinoquinazoline derivatives as potent inhibitors of epidermal growth factor receptor with antitumor activity

Two new series of new compounds containing a 6-amino-substituted group or 6-acrylamide-substituted group linked to a 4-anilinoquinazoline nucleus have been discovered as potential EGFR inhibitors. These compounds proved efficient effects on antiproliferative activity and EGFR–TK inhibitory activity. Especially, N⁶-((5-bromothiophen-2-yl)methyl)-N⁴-(3-chlorophenyl)quinazoline-4,6-diamine (5e), showed the most potent inhibitory activity (IC₅₀ = 3.11 μM for Hep G2, IC₅₀ = 0.82 μM for A549). The EGFR molecular docking model suggested that the new compound is nicely bound to the region of EGFR, and cell morphology by Hoechst stain experiment suggested that these compounds efficiently induced apoptosis of A549 cells.     

Higher somatic cells counted by the electronic counter method do not influence renneting properties of goat milk

Laws of different countries regarding SCC of goat milk are not in agreement with each other and sometimes they fix a threshold for the enhancement of dairy products. The aim of this study was to assess if renneting properties of goat milk are influenced by higher somatic cell count (SCC) measured by an electronic cell counter. Milk samples, taken throughout the lactation of 169 goats from three farms, were analyzed for chemical, physical, hygienic and renneting properties. Samples were divided into three levels on the basis of their SCC: L: low level, samples with SCC lower than 10⁶ cells/ml; M: medium, between 10⁶ and 2 × 10⁶ cells/ml; H: high, higher than 2 × 10⁶ cells/ml. Milk clotting time was between 12.07 and 13.31 min, curd firming time between 1.68 and 2.05 min and curd firmness between 41.66 and 48.97 mm. All the three renneting properties were not affected by the SCC level but they were highly correlated with other factors as protein content and pH. Furthermore, the microbial count showed a high positive correlation with SCC. These results showed that in goat milk, contrarily to other dairy species, higher SCC did not affect renneting properties and that counting of somatic cells by using electronic cell counters might be not suitable for the improvement of dairy products.     

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.     

Identification and evaluation of apoptotic compounds from Garcinia paucinervis

Four new compounds, paucinervins A–D (1–4), and 15 known ones were isolated from the leaves of Garcinia paucinervis. The structures of the new compounds were elucidated by spectroscopic evidences. All of the 19 compounds were evaluated for their apoptosis-inducing effects using HeLa-C3 cells which have been genetically engineered to possess a fluorescent biosensor capable of detecting caspase-3 activation. Eight of them were found to activate caspase-3 in HeLa-C3 cells within 72 h at the concentration of 25 μM. Moreover, the values of IC₅₀ were measured for all four new compounds on HeLa cells using the MTT assay. Among them, compound 2 (paucinervin B) had the lowest IC₅₀ value of 9.5 μM, while the other three new compounds had much higher IC₅₀ values of 29.5, 52.5, and 95.6 μM, respectively. This result shows that paucinervin B has the strongest inhibitory effect against HeLa cell growth among these four newly identified paucinervins and it may have the potential to be developed into a new anticancer candidate.     

Discovery of substituted benzyl tetrazoles as histamine H3 receptor antagonists

A series of potent and subtype selective H3 receptor antagonists containing a novel tetrazole core and diamine motif is reported. A one-pot multi-component Ugi reaction was utilised to rapidly develop the structure–activity relationships (SAR) of these compounds. Optimisation for liver microsome stability (t_1/2 >60 min), minimal CYP inhibition (IC₅₀ >50 μM) and high cell permeability (Caco-2 P_app >20 × 10^?6 cm/s) identified several compounds with drug-like properties.     

Design,synthesis and biological evaluation of novel 4-anilinoquinazolines with C-6 urea-linked side chains as inhibitors of the epidermal growth factor receptor

A novel series of anilinoquinazoline compounds with C-6 urea-linked side chains was designed and synthesized as reversible inhibitors of epidermal growth factor receptor (EGFR) based on the structure–activity relationships (SARs) of anilinoquinazoline inhibitors. All compounds demonstrated good inhibition of EGFR wild type (EGFR wt) (IC₅₀ = 0.024–1.715 μM) and inhibited proliferation of A431cell line (IC₅₀ = 0.116–22.008 μM). The binding mode of compounds 8a, 8d, 8k and 8o was consistent with the biological results. Moreover, compounds 8k and 8l almost completely blocked the phosphorylation of EGFR in A431 cell line at 0.01 μM. Interestingly, all of the compounds also demonstrated moderate inhibition of EGFR/T790M/L858R (IC₅₀ = 0.049–5.578 μM). In addition, compounds 8f and 8h blocked the autophosphorylation of EGFR in NCI-H1975 cells at high concentration (10 μM), and compound 8f was confirmed to be an irreversible inhibitor through the dilution method. Importantly, the compounds with C-6 urea-linked side chains which did not contain Michael acceptors demonstrated moderate to strong irreversible EGFR inhibition.     

Suitability of a cytotoxicity assay for detection of potentially harmful compounds produced by freshwater bloom-forming algae

Detecting harmful bioactive compounds produced by bloom-forming pelagic algae is important to assess potential risks to public health. We investigated the application of a cell-based bioassay: the rainbow trout gill-w1 cytotoxicity assay (RCA) that detects changes in cell metabolism. The RCA was used to evaluate the cytotoxic effects of (1) six natural freshwater lake samples from cyanobacteria-rich lakes in central Ontario, Canada; (2) analytical standards of toxins and noxious compounds likely to be produced by the algal communities in these lakes; and (3) complex mixtures of compounds produced by cyanobacterial and chrysophyte cultures. RCA provided a measure of lake water toxicity that could not be reproduced using toxin or noxious compound standards. RCA was not sensitive to toxins and only sensitive to noxious compounds at concentrations higher than reported environmental averages (EC₅₀ ≥ 10³ nM). Cultured algae produced bioactive compounds that had recognizable dose dependent and toxic effects as indicated by RCA. Toxicity of these bioactive compounds depended on taxa (cyanobacteria, not chrysophytes), growth stage (stationary phase more toxic than exponential phase), location (intracellular more toxic than extracellular) and iron status (cells in high-iron treatment more toxic than cells in low-iron treatment). The RCA provides a new avenue of exploration and potential for the detection of natural lake algal toxic and noxious compounds.     

Evaluation of zinc tolerance and accumulation potential of the coastal shrub Limoniastrum monopetalum (L.) Boiss.

The coastal shrub Limoniastrum monopetalum is capable of growth in soil containing extremely high concentrations of heavy metals. A greenhouse experiment was conducted in order to investigate the effects of a range of Zn concentrations (0–130 mmol l⁻¹) on growth and photosynthetic performance, by measuring relative growth rate, total leaf area, plant height, gas exchange, chlorophyll fluorescence parameters and photosynthetic pigment concentrations. We also determined the total zinc, nitrogen, phosphorus, sulphur, calcium, magnesium, sodium, potassium, iron and copper concentrations in the plant tissues. The study species demonstrated hypertolerance to Zn stress, since survival was recorded with leaf concentrations of up to 1700 mg Zn kg⁻¹ dry mass when treated with 130 mmol Zn l⁻¹. L. monopetalum exhibited little overall effects on photosynthetic function at Zn levels of up to 90 mmol l⁻¹. At greater external Zn concentration, plant growth was negatively affected, due in all probability to the recorded decline in net photosynthetic rate, which may be linked to the adverse effect of the metal on photosynthetic electron transport. Growth parameters were virtually unaffected by leaf tissue concentrations as high as 1400 mg Zn kg⁻¹ dry mass thus indicating that this species could play an important role in the phytoremediation of Zn-polluted areas.