The neutral comet assay is sufficient to identify an apoptotic ‘window’ by visual inspection

As a sensitive technique for measuring DNA damage, the alkaline comet assay (capable of detecting and distinguishing apoptotic and necrotic damage) shows significantly greater ability to detect DNA breaks than a neutral counterpart. Using a heat shock model, we show that the fraction of visually detectable comets decreases using the neutral assay as cell death shifts from apoptosis to necrosis. We also show a virtual absence of neutral comets in cells dying by necrosis in another model. We conclude that the non-denaturing assay allows identification of putative apoptotic windows by showing sensitivity to apoptotic, but not necrotic, DNA damage.     

Fluorescence as an alternative to light-scatter gating strategies to identify frozen–thawed cells with flow cytometry

Flow cytometry is a key instrument in biological studies, used to identify and analyze cells in suspension. The identification of cells from debris is commonly based on light scatter properties as it has been shown that there is a relationship between forward scattered light and cell volume and this has become common practice in flow cytometry. Cryobiological conditions induce changes in cells that alter their light scatter properties. Cells with membrane damage from freeze–thaw stress produce lower forward scatter signals and may fall below standard forward scatter thresholds. In contrast to light scatter properties that cannot identify damaged cells from debris, fluorescent dyes used in membrane integrity and mitochondrial polarization assays are capable of labeling and discriminating all cells in suspension. Under cryobiological conditions, isolating cell populations is more effectively accomplished by gating on fluorescence rather than light scatter properties. This study shows the limitations of using forward scatter thresholds in flow cytometry to identify and gate cells after exposure to a freeze–thaw protocol and demonstrates the use of fluorescence as an alternative means of identifying and analyzing cells.     

Simple assay for adsorption of proteins to the air–water interface

A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air–water interface (AWI) of aqueous samples. This assay requires no more than a 20 µL aliquot of sample, at a protein concentration of no more than1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI. Use of this assay is suggested as a way to pre-screen the effect of various additives and chemical modifications that one might use to optimize the preparation of grids, although the final proof of optimization still requires further screening of grids in the electron microscope. In those cases when the assay establishes that a given protein does form a sacrificial, denatured-protein monolayer, it is suggested that subsequent optimization strategies might focus on discovering how to improve the adsorption of native proteins onto that monolayer, rather than to prevent its formation. A second alternative might be to bind such proteins to the surface of rationally designed affinity grids, in order to prevent their diffusion to, and unwanted interaction with, the AWI.     

Genetic viability and population history of the giant panda, putting an end to the "evolutionary dead end"?

The giant panda (Ailuropoda melanoleuca) is currently threatened by habitat loss, fragmentation, and human persecution. Its dietary specialization, habitat isolation, and reproductive constraints have led to a perception that this is a species at an "evolutionary dead end," destined for deterministic extinction in the modern world. Here we examine this perception by a comprehensive investigation of its genetic diversity, population structure, and demographic history across its geographic range. We present analysis of 655 base pairs of mitochondrial (mt) control region (CR) DNA and 10 microsatellite loci for samples from its 5 extant mountain populations (Qinling, Minshan, Qionglai, Liangshan, and Lesser Xiangling). Surprisingly, extant populations display average to high levels of CR and microsatellite diversity compared with other bear species. Genetic differentiation among populations was significant in most cases but was markedly higher between Qinling and the other mountain ranges, suggesting, minimally, that the Qinling population should comprise a separate management unit for conservation purposes. Recent demographic inference using microsatellite markers demonstrated a clear genetic signature for population decline starting several thousands years ago or even further back in the past, and being accelerated and enhanced by the expansion of human populations. Importantly, these data suggest that the panda is not a species at an evolutionary "dead end," but in common with other large carnivores, has suffered demographically at the hands of human pressure. Conservation strategies should therefore focus on the restoration and protection of wild habitat and the maintenance of the currently substantial regional genetic diversity, through active management of disconnected populations.     

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2β interaction

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2β in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α^1–335 and the fluorescent probe CF-Ahx-Pc as a CK2β analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α^1–335. Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2β. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α^1–335 complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α^1–335 were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a K_D value of 240 nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2β, with an IC₅₀ value of 92 μM.     

Combined use of radioimagers and radioactive 3′OH DNA nick end labelling to quantify apoptosis in cell lines and tissue sections: applications to virus-induced apoptosis

DNA fragmentation is a key feature of the degradation phase of apoptosis. In this work we have developed an assay, based on radioimager (-IMAGER and -IMAGER) quantification of radioactive nick end labelling (RANEL), which is quantitative, rapid and sensitive to study in vitro and in vivo induced apoptosis. To establish the technique, in vitro apoptosis of T cell lines was induced by stimulation of the Fas receptor; cells were labelled using TdT-mediated [-³³P] dCTP nick end labelling, after which then radioactivity was quantified using a -IMAGER. We have also shown that the RANEL method can be applied to the quantification and visualisation, by -IMAGER analysis, of liver tissue sections from mouse Fas-induced fulminant hepatitis or from Dengue-1 virus infected individuals. Finally, this system has also been used to detect apoptosis induced by rabies virus in Jurkat T cells. These data have established a large field of application for the RANEL assay.     

On the use of one-step perturbation to investigate the dependence of NOE-derived atom–atom distance bound violations of peptides upon a variation of force-field parameters

The method of one-step perturbation can be used to predict from a single molecular dynamics simulation the values of observable quantities as functions of variations in the parameters of the Hamiltonian or biomolecular force field used in the simulation. The method is used to predict violations of nuclear overhauser effect (NOE) distance bounds measured in nuclear magnetic resonance (NMR) experiments by atom–atom distances of the NOE atom pairs when varying force-field parameters. Predictions of NOE distance bound violations between different versions of the GROMOS force field for a hexa-β-peptide in solution show that the technique works for rather large force-field parameter changes as well as for very different NOE bound violation patterns. The effect of changing individual force-field parameters on the NOE distance bound violations of the β-peptide and an α-peptide was investigated too. One-step perturbation, which in this case is equivalent to reweighting configurations, constitutes an efficient technique to predict many values of different quantities from a single conformational ensemble for a particular system, which makes it a powerful force-field development technique that easily reduces the number of required separate simulations by an order of magnitude.     

Oligomerization–function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor–ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag–probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~ 10%), whereas ~ 70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20 s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90 s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~ 1 nM) was lower than that for half-maximum autophosphorylation (~ 8 nM), which suggested the presence of an inactive dimer binding a single EGF molecule.     

Implementation of a dose–response curve for γ-radiation in the Portuguese population by use of the chromosomal aberration assay

An in vitro dose–response curve following exposure to γ-radiation was determined at the IST/ITN, by use of the chromosomal aberration assay. This is the first study of this kind carried out among the Portuguese population. Un-irradiated and γ-irradiated peripheral blood lymphocytes from 16 healthy donors were cultured. A total of 22,395 metaphases were analyzed for frequency and distribution of dicentrics and centric rings, as a function of the radiation dose. The dose–response data for dicentrics and dicentrics plus centric rings were fitted by use of a linear–quadratic model: Y_dic = (0.0011 ± 0.0006) + (0.0105 ± 0.0035)D + (0.0480 ± 0.0019)D² and Y_{dic + rings} = (0.0011 ± 0.0006) + (0.0095 ± 0.0036)D + (0.0536 ± 0.0020)D². Also, calibration curves related to age and gender were determined, but no significant differences were found. Following the establishment of the dose–response curves, a validation experiment was carried out with three individuals. Real and estimated doses, obtained with the dose–response curves, were in agreement. These results give us confidence to apply both dose–response calibration curves in future biological dosimetry requirements.     

Development of an LC–MS/MS assay to determine plasma pharmacokinetics of the radioprotectant octadecyl thiophosphate (OTP) in monkeys

Octadecenyl thiophosphate (OTP), a synthetic analogue of the lysophospholipid growth factor lysophosphatidic acid (LPA), significantly reduces mortality following a lethal dose of LD_80/30 radiation exposure in a mouse model of whole-body irradiation. To facilitate dose scaling between species, we developed a novel liquid chromatography/tandem mass spectrometry (LC–MS/MS) for the preclinical pharmacokinetic characterization of OTP in monkeys. Sample extraction was carried out using a butanol based liquid–liquid extraction method. A partially deuterated OTP analogue was used as internal standard (IS). OTP and IS were separated by reversed-phase liquid chromatography on a C-8 column using 10 mM ammonium acetate and acetonitrile. A triple quadrupole mass spectrometer operating in the negative electrospray ionization mode with multiple reaction monitoring was used to detect OTP and IS transitions of m/z 363.1 → 95.0 and 403.1 → 95.0. The method was applied to determine pharmacokinetic parameters in monkeys receiving a single oral OTP dose (3 mg/kg). OTP is readily absorbed with a relatively long half-life which supports further preclinical testing of OTP as a radioprotectant in monkeys.     

Kynostatin and 17β-estradiol prevent the apoptotic death of human neuroblastoma cells exposed to HIV-1 protease

A significant number of adult male patients with acquired immunodeficiency syndrome develop cerebral atrophy and progressive brain disorders such as dementia complex and neuropsychiatric problems. Upon entering the brain via activated macrophages or microglias, the human immunodeficiency type 1 virus (HIV-1) may produce cytotoxic factors such as HIV-1 envelope protein (gp 120) and protease. Owing to significant proteolysis of nonviral proteins, the protease derived from HIV-1 may be detrimental to brain cells and neurons. Our results revealed that HIV-1 protease, at nanomolar concentrations, was as potent as gp 120 in causing neurotoxicity in human neuroblastoma neurotypic SH-SY5Y cells. As shown by the Oncor ApopTag staining procedure, HIV-1 protease significantly increased the number of apoptotic cells over the serum-free controls. Moreover, HIV-1 protease-induced neurotoxicity was blocked by a selective protease inhibitor, kynostatin (KNI-272). Antioxidants such as 17-estradiol, melatonin, andS-nitrosoglutathione also prevented protease-induced neurotoxicity. These findings indicate that oxidative proteolysis may mediate HIV-1 protease-induced apoptosis and the degeneration of neurons and other brain cells. Centrally active protease inhibitors and antioxidants may play an important role in preventing cerebral atrophy and associated dementia complex caused by HIV-1.     

The use of ultrasound to identify milk ejection in women – tips and pitfalls

Background

Rates of exclusive breastfeeding in China are relatively low and below national targets. The aim of this study was to document the factors that influence exclusive breastfeeding initiation in Zhejiang, PR China.

Methods

A cohort study of infant feeding practices was undertaken in Zhejiang Province, an eastern coastal region of China. A total of 1520 mothers who delivered in four hospitals located in city, suburb and rural areas during late 2004 to 2005 were enrolled in the study. Multivariate logistic regression analysis was used to explore factors related to exclusive breastfeeding initiation.

Results

On discharge from hospital, 50.3% of the mothers were exclusively breastfeeding their infants out of 96.9% of the mothers who had earlier initiated breastfeeding. Exclusive breastfeeding was positively related to vaginal birth, baby's first feed being breast milk, mother living in the suburbs or rural areas, younger age of mother, lower maternal education level and family income.

Conclusion

The exclusive breastfeeding rate in Zhejiang is only 50.3% on discharge and does not reach Chinese or international targets. A number of behaviours have been identified in the study that could be potentially incorporated into health promotion activities.     

Effect of deletions 5′ to the translation initiation sequence on the expression of an mRNA in animal cells

To learn if an mRNA·18S rRNA interaction or a special secondary structure in the mRNA start region is essential for translation in eukaryotic cells, we constructed recombinant plasmids with the SV40 early promoter 5 to part of the Escherichia coli tuf B-lacZ gene. Deletion of bases potentially complementary to the 18S rRNA highly increased the transient -galactosidase expressed in transfected CHO cells. Deletion of bases that fostered formation of potential hairpins with the mRNA 5-terminus or altered the structure of the coding region reduced -galactosidase activity suggesting that these features of the mRNA secondary structure may be essential for initiation of translation. Computer aided analysis of the potential structure of 290 mRNAs suggests these are conserved features of the initiation region.     

Reaction modelling and simulation to assess the integrated use of transketolase and ω-transaminase for the synthesis of an aminotriol

The most attractive, as well as challenging, multistep organic syntheses would preferably be carried out in a single reactor, as a one-pot synthesis. For biocatalytic syntheses, multistep reactions in one-pot mode bring a number of advantages, while at the same time raising unique challenges such as the compatibility of different biocatalysts. In this paper, we have developed a transketolase–transaminase (TK-TAm) two-step one-pot aminotriol synthesis reaction model, which integrates reaction kinetic models with process characterization (consisting of component degradation as a function of pH and concentration, aldehyde toxicity towards the enzyme, and ketol donor and acceptor side-reactions with TAm). Based on the analysis of the effect of the TAm/TK activity ratio on product yield, simulations provided guidance for further process and biocatalyst development.     

Differential location of α-expansin proteins during the accommodation of root cells to an arbuscular mycorrhizal fungus

-Expansins are extracellular proteins that increase plant cell-wall extensibility. We analysed their pattern of expression in cucumber roots in the presence and in the absence of the mycorrhizal fungus, Glomus versiforme. The distribution of -expansins was investigated by use of two polyclonal antibodies (anti-EXPA1 and anti-EXPA2, prepared against two different cucumber -expansins) in immunoblotting, immunofluorescence, and immunogold experiments. Immunoblot results indicate the presence of a 30-kDa band specific for mycorrhizal roots. The two antibodies identify antigens with a different distribution in mycorrhizal roots: anti-EXPA1 labels the interface zone, but the plant cell walls only weakly. By contrast, the anti-EXPA2 labels only the plant cell walls. In order to understand the potential role of -expansins during the accommodation of the fungus inside root cells, we prepared semi-thin sections to measure the size of cortical cells and the thickness of cortical cell walls in mycorrhizal and non-mycorrhizal root. Mycorrhizal cortical cells were significantly larger than non-mycorrhizal cells and had thicker cell walls. In double-labelling experiments with cellobiohydrolase–gold complex, we observed that cellulose was co-localized with -expansins. Taken together, the results demonstrate that -expansins are more abundant in the cucumber cell walls upon mycorrhizal infection; we propose that these wall-loosening proteins are directly involved in the accommodation of the fungus by infected cortical cells.     

On the appropriateness of use of a continuous formulation for the modelling of discrete multireactant systems following Michaëlis–Menten kinetics

The possibility of solving the mass balances to a multiplicity of substrates within a CSTR in the presence of a chemical reaction following Michaelis-Menten kinetics using the assumption that the discrete distribution of said substrates is well approximated by an equivalent continuous distribution on the molecular weight is explored. The applicability of such reasoning is tested with a convenient numerical example. In addition to providing the limiting behavior of the discrete formulation as the number of homologous substrates increases, the continuous formulation yields in general simpler functional forms for the final distribution of substrates than the discrete counterpart due to the recursive nature of the solution in the latter case.List of Symbols C{N. M} mol/m³ concentration of substrate containing N monomer residues each with molecular weight M - {N, M} normalized value of C{N. M} - C {M} mol/m³ da concentration of substrate of molecular weight M - _in normalized value of C {M} at the i-th iteration of a finite difference method - {M} normalized value of C {M} - C ₀{N.M} mol/m³ inlet concentration of substrate containing N monomer residues each with molecular weight M - {N ·M} normalized value of C₀{N. M} - _{0 i} normalized value of C ₀ {M} at the i-th iteration of a finite difference method - C ₀ {M} mol/m³ da initial concentration of substrate of molecular weight M - C _tot mol/m³ (constant) overall concentration of substrates (discrete model) - C _tot mol/m³ (constant) overall concentration of substrates (continuous model) - D deviation of the continuous approach relative to the discrete approach - i dummy integer variable - I arbitrary integration constant - j dummy integer variable - k dummy integer variable - K _m mol/m³ Michaëlis-Menten constant for the substrates - l dummy integer variable - M da molecular weight of substrate - M normalized value of M - M da maximum molecular weight of a reacting substrate - N number of monomer residues of a reacting substrate - N maximum number of monomer residues of a reacting substrate - N total number of increments for the finite difference method - Q m³/s volumetric flow rate of liquid through the reactor - S inert product molecule - S _i substrate containing i monomer residues - V m³ volume of the reactor - v _max mol/m³ s reaction rate under saturating conditions of the enzyme active site with substrate - v _max{N. M} mol/m³ s reaction rate under saturating conditions of the enzyme active site with substrate containing N monomer residues with molecular weight M - _max{N · M} dimensionless value of v_max{N. M} (discrete model) - _max{M} dimensionless value of v _max {M} (continuous model) - mol/m³ s molecular weight-averaged value of v_max (discrete model) - mol.da/m³s molecular weight-averaged value of v_max (continuous model) - v _max {M} mol.da/m³s reaction rate under saturating conditions of the enzyme active site with substrate with molecular weight M - _max {M} dimensionless value of v_max{M} - _{max, (i)} dimensionless value of v_max{M} at the i-th iteration of a finite difference method - v _max mol/m³ s reference constant value of v _max Greek Symbols dimensionless operating parameter (discrete distribution) - dimensionless operating parameter (continuous distribution) - M da (average) molecular weight of a monomeric subunit - M selected increment for the finite difference method - auxiliary corrective factor (discrete model)     

Alanine Scanning Mutagenesis Identifies an Asparagine–Arginine–Lysine Triad Essential to Assembly of the Shell of the Pdu Microcompartment

Bacterial microcompartments (MCPs) are the simplest organelles known. They function to enhance metabolic pathways by confining several related enzymes inside an all-protein envelope called the shell. In this study, we investigated the factors that govern MCP assembly by performing scanning mutagenesis on the surface residues of PduA, a major shell protein of the MCP used for 1,2-propanediol degradation. Biochemical, genetic, and structural analysis of 20 mutants allowed us to determine that PduA K26, N29, and R79 are crucial residues that stabilize the shell of the 1,2-propanediol MCP. In addition, we identify two PduA mutants (K37A and K55A) that impair MCP function most likely by altering the permeability of its protein shell. These are the first studies to examine the phenotypic effects of shell protein structural mutations in an MCP system. The findings reported here may be applicable to engineering protein containers with improved stability for biotechnology applications.