Causes of shear sensitivity of the toxic dinoflagellate Protoceratium reticulatum

Dinoflagellates have proven extremely difficult to culture because they are inhibited by low‐level shear forces. Specific growth rate of the toxic dinoflagellate Protoceratium reticulatum was greatly decreased compared with static control culture by intermittent exposure to a turbulent hydrodynamic environment with a bulk average shear rate that was as low as 0.3 s^?1. Hydrodynamic forces appeared to induce the production of reactive oxygen species (ROS) within the cells and this caused peroxidation of cellular lipids and ultimately cell damage. Exposure to damaging levels of shear rate correlated with the elevated level of lipoperoxides in the cells, but ROS levels measured directly by flow cytometry did not correlate with shear induced cell damage. This was apparently because the measured level of ROS could not distinguish between the ROS that are normally generated by photosynthesis and the additional ROS produced as a consequence of hydrodynamic shear forces. Continuously subjecting the cells to a bulk average shear rate value of about 0.3 s^?1 for 24‐h caused an elevation in the levels of chlorophyll a, peridinin and dinoxanthin, as the cells apparently attempted to counter the damaging effects of shear fields by producing pigments that are potential antioxidants. In static culture, limitation of carbon dioxide produced a small but measureable increase in ROS. The addition of ascorbic acid (0.1 mM) to the culture medium resulted in a significant protective effect on lipid peroxidation, allowing cells to grow under damaging levels of shear rates. This confirmed the use of antioxidant additives as an efficient strategy to counter the damaging effects of turbulence in photobioreactors where shear sensitive dinoflagellates are cultivated. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Kinetics of transport and phosphorylation of glucose in cancer cells

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS‐30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D ‐[2‐³H‐glucose] as physiological substrate. Kinetic analysis revealed two components at low‐ and high‐glucose concentrations in both tumor cells. At low glucose and 37°C, the V_max was 55 ± 20 and 17.2 ± 6 nmol (min × mg protein)^?1, whereas the K_m was 0.52 ± 0.7 and 9.3 ± 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC₅₀ = 0.44 ± 0.1; K_i = 0.3 ± 0.1 µM) and phloretin (IC₅₀ = 8.7 µM) in AS‐30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS‐30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS‐30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS‐30D carcinoma and HeLa cells. The much lower GLUT V_max and catalytic efficiency (V_max/K_m) values in comparison to those of G6P‐sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target. J. Cell. Physiol. 221: 552–559, 2009. © 2009 Wiley‐Liss, Inc.     

Genotoxic activity of halogenated phenylglycine derivatives

The discovery of genotoxic amino acids derived from phenylglycine, and possessing halogen substituents, is described. The utility of hypervalent iodine reagents in the synthesis of this class of compounds is highlighted. The mechanism of action of the (haloaryl)glycines was studied in Saccharomyces cerevisiae.     

Effect of day 3 embryo morphometrics and morphokinetics on survival and implantation after slow freezing-thawing and after vitrification-warming: a retrospective cohort study

Background

Morphometric and morphokinetic evaluation of in vitro cultured human embryos allows evaluation without time restriction and reduces intra- and inter-observer variability. Even though these technologies have been reported to improve the quality of cleavage stage embryo evaluation during fresh culture, possible advantages in the evaluation of cryopreserved embryos have been scarcely explored. This study aims to compare morphometric and morphokinetic parameters between slow frozen and vitrified embryos and to determine their relationship to embryo survival and implantation rate (IR) after thawing/warming.

Methods

During fresh culture, morphometric characteristics (Total Cell Volume (TCV), symmetry, fragmentation and number of blastomeres) were measured in 286 thawed/warmed embryos. Likewise, after thawing/warming, similar morphometric characteristics were measured in 135 survived embryos. Moreover, morphokinetic parameters (time to mitosis resumption and time to compaction) were measured in 90 embryos after thawing/warming. Then, using linear regression, we investigated the differences between vitrified and slow frozen embryos and the relation of the measured characteristics to embryo survival and IR. Statistical corrections were applied to account for data clustering and for multiple testing.

Results

Vitrified embryos resume mitosis and start compaction significantly earlier than slow frozen embryos. Mitosis resumption rate was 82% for vitrified and 63% for slow frozen embryos and median time to mitosis resumption was 7.6 h and 13.1 h (p = 0.02), respectively. Compaction rate was 62% in vitrified and only 23% in slow frozen embryos. Median time to compaction was 18.1 h for vitrified embryos but, for slow frozen could not be computed since less than half of the slow frozen embryos reached compaction (p = 0.0001). Moreover, intact embryos resume mitosis significantly earlier than not intact ones regardless of the freezing method (rate: 79% vs. 66%, median time: 7.6 h vs 14.6 h, respectively, p = 0.03). Regarding morphometrics, slow frozen embryos showed lower TCV and higher blastomere symmetry after thawing than vitrified embryos despite having similar blastomere number. IR was related to blastomere number at cryopreservation in slow frozen embryos, but not in vitrified ones.

Conclusions

Interestingly, vitrified/warmed embryos undergo mitosis resumption and compaction significantly earlier than slow frozen/thawed embryos. However, the clinical use of this morphokinetic parameters still remains to be investigated in larger studies.

Trial registration

Retrospectively registered on December 15, 2015 NCT02639715.

     

Cell Adhesion and Proliferation on Sulfonated and Non-Modified Chitosan Films

Three types of chitosan-based films have been prepared and evaluated: a non-modified chitosan film bearing cationizable aliphatic amines and two films made of N-sulfopropyl chitosan derivatives bearing both aliphatic amines and negative sulfonate groups at different ratios. Cell adhesion and proliferation on chitosan films of C2C12 pre-myoblastic cells and B16 cells as tumoral model have been tested. A differential cell behavior has been observed on chitosan films due to their different surface modification. B16 cells have shown lower vinculin expression when cultured on sulfonated chitosan films. This study shows how the interaction among cells and material surface can be modulated by physicochemical characteristics of the biomaterial surface, altering tumoral cell adhesion and proliferation processes.     

Short-term effects of litter from 21 woody species on plant growth and root development

Background and aims

Plant litter has an important role in terrestrial ecosystems (Lambers et al. 2008). Our aim was to assess the short-term effect of litter from 21 woody species (deciduous and evergreens) on plant growth and root development.

Methods

We conducted a short-term experiment (10 weeks) under controlled conditions adding litter from 21 woody species to pots with Dactylis glomerata (target species). We determined plant biomass and root development and related these variables to decomposition rate and litter quality.

Results

Litter from two species enhanced plant growth whereas litter of five species inhibited it. Considering all species in the data set, plant growth was associated to litter with high decomposition rate and high litter quality: high Ca and N concentration and low polyphenols concentration. However, excluding from the analyses the two species that increased growth, litter inhibition effect on plant growth was related to the litter-polyphenols concentration. Plants growing with nutrient-richer litter had a lower proportion of fine roots which could be related to a litter mediated increase in soil nutrient.

Conclusions

Enhanced plant growth or, on the contrary, plant growth inhibition could be the result of a positive or, in turn, negative balance between nutrient and polyphenols concentration in litter.     

A collection of open source applications for mass spectrometry data mining

We present several bioinformatics applications for the identification and quantification of phosphoproteome components by MS. These applications include a front‐end graphical user interface that combines several Thermo RAW formats to MASCOT? Generic Format extractors (EasierMgf), two graphical user interfaces for search engines OMSSA and SEQUEST (OmssaGui and SequestGui), and three applications, one for the management of databases in FASTA format (FastaTools), another for the integration of search results from up to three search engines (Integrator), and another one for the visualization of mass spectra and their corresponding database search results (JsonVisor). These applications were developed to solve some of the common problems found in proteomic and phosphoproteomic data analysis and were integrated in the workflow for data processing and feeding on our LymPHOS database. Applications were designed modularly and can be used standalone. These tools are written in Perl and Python programming languages and are supported on Windows platforms. They are all released under an Open Source Software license and can be freely downloaded from our software repository hosted at GoogleCode.