Enhanced mitochondrial G-quadruplex formation impedes replication fork progression leading to mtDNA loss in human cells.

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.

Magnetic interactions in graphene decorated with iron oxide nanoparticles.

We present the studies of structural and magnetic properties of graphene composites prepared with several quantities ofα-Fe2O3dopant of 5%, 25% and 50% made with either ethanol or acetone. Our studies showed the presence of a weak magnetic order up to room temperature and saturation magnetization close to 0.2 emu g-1in pure commercial graphene. With regard to magnetic properties of our graphene + iron oxide samples, the solvent used during the preparation of the composite had a significant influence on them. For graphene + Fe2O3samples made with acetone the magnetic properties of pure graphene played a major role in the overall magnetic susceptibility and magnetization. On the other hand, for graphene + iron oxide samples made with ethanol we observed the presence of superparamagnetic blocking atT < 110 K which was due to the additional appearance ofγ-Fe3O4nanoparticles. Changes in the synthesis solvent played a major role in the magnetic properties of our graphene + Fe2O3nanocomposite samples resulting in much higher saturation magnetization for the samples made with ethanol. Both the shape and the parameters characterizing magnetization hysteresis loops depend strongly on the amount of iron oxide and changes in the preparation method.

LY3041658/ interleukin-8 complex structure as targets for IL-8 small molecule inhibitors discovery using a combination of in silico methods.

Since interleukin-8 (IL-8/CXCL8) and its receptor, CXCR1 and CXCR2, were known in the early 1990s, biological pathways related to these proteins were proven to have high clinical value in cancer and inflammatory/autoimmune conditions treatment. Recently, IL-8 has been identified as biomarker for severe COVID-19 patients and COVID-19 prognosis. Boyles et al. (mAbs 12 (2020), pp. 1831880) have published a high-resolution X-ray crystal structure of the LY3041658 Fab in a complex human CXCL8. They described the ability to bind to IL-8 and the blocking of IL-8/its receptors interaction by the LY3041658 monoclonal antibody. Therefore, the study has been designed to identify potential small molecules inhibiting interleukin-8 by targeting LY3041658/IL-8 complex structure using an in silico approach. A structure­based pharmacophore and molecular docking models of the protein active site cavity were generated to identify possible candidates, followed by virtual screening with the ZINC database. ADME analysis of hit compounds was also conducted. Molecular dynamics simulations were then performed to survey the behaviour and stability of the ligand-protein complexes. Furthermore, the MM/PBSA technique has been utilized to evaluate the free binding energy. The final data confirmed that one newly obtained compound, ZINC21882765, may serve as the best potential inhibitor for IL-8.

Discovery of superconductivity in AlB2-type hexagonal YGa2.

A crystalline sample YGa2with the AlB2-type hexagonal structure has been synthesized using the self-flux method. We measured electrical resistivity and specific heat down to 0.4 K and in fields up to 200 mT. The obtained data reveal type-II superconductivity. Unusual behaviour of the temperature dependence of upper critical fieldHc2(Tc) was attributed to the crystal twinning of two phases possessing close lattice parameters. We determined thermodynamic parameters in the superconducting and normal states of YGa2. DFT calculations indicated a large change in the Fermi velocity of carriers nearby cylinder-like structured Fermi surface along the Γ -Aline.

Physical properties of the layered oxypnictide Sr2ScFeAsO3: a Mössbauer study down to 1.7 K.

A polycrystalline sample of Sr2ScFeAsO3 was studied by 57Fe Mössbauer spectroscopy down to 1.7 K. In contrast to the earlier Mössbauer data, the obtained in this work results indicate that Sr2ScFeAsO3 is in paramagnetic state down to 10 K, while the spectra recorded at 4.6 K and 1.7 K show a weak magnetic order of Fe moments in the Fe2As2 layers. Temperature dependences of isomer shift and quadrupole splitting/shift are compared with specific heat and electrical resistivity data from earlier investigations revealing different local Debye temperatures for the Fe2As2 and perovskite-related Sr2ScO3 layers. Finally, a fast decrease of the carrier density was observed below 80 K and this effect seems to be responsible for the absence of superconductivity in the studied compound.

57Fe Mössbauer and magnetic studies of Th7Co2.5 57Fe0.5 and Th7Ni2.5 57Fe0.5 superconductors.

The noncentrosymmetric Th7Co2.5 57Fe0.5 and Th7Ni2.5 57Fe0.5 superconductors were studied by means of 57Fe transmission Mössbauer spectroscopy (TMS), ac-magnetic susceptibility and magnetization measurements. The low-temperature data showed that both compounds are type-II superconductors with transition temperature close to [Formula: see text] K. The collected Mössbauer spectra of the studied samples consist of one well-resolved doublet over the temperature range measured. Neither abnormal behaviour of the hyperfine parameters at or near [Formula: see text] nor phonon softening were found. A comparison of 57Fe Mössbauer data of Ni- and Co-based superconductors with those of Th7Fe3 indicated that with decreasing 3d-electron numbers across the series Th7Ni2.5 57Fe0.5 [Formula: see text] Th7Co2.5 57Fe0.5 [Formula: see text] Th7Fe3, the s-electron density around the Fe nucleus and as well as the electric field gradient strength increases. The latter finding is associated with increasing asymmetricity of 3d-electron distribution and this fact presumably gives arise to an exotic superconductivity in the Co- and Fe-based compounds.

Superconducting-state properties and electronic band structure calculations of a noncentrosymmetric Th7Ni3 compound.

Superconducting-state properties of a noncentrosymmetric Th7Ni3 compound have been investigated using magnetic, electrical resistivity and specific heat measurements as well as by electronic band structure calculations. The study reveals that the studied compound is a dirty type-II superconductor with [Formula: see text] K and a weak electron-phonon coupling [Formula: see text]. Moreover, in contrast to an exotic superconductivity observed previously in Th7Fe3 and Th7Co3, data reported in this paper give clear evidence that Th7Ni3 is a conventional single-gap superconductor. The experimental results are supported by DFT calculations of the electronic band structure, density of states, electron localization functions and Fermi surfaces which were performed using the full-potential linear muffin-tin-orbital and full-potential linearized augmented plane wave methods. Theoretical data show that asymmetric spin-orbit coupling in Th7Ni3 is quite small; hence the electronic band structure of this compound is weakly affected by the spin-orbit effects. We have determined fundamental parameters of Th7Ni3 and compared the superconducting properties with other Th7T3 (T = Fe, Co and Ru) compounds.

Specific heat, Electrical resistivity and Electronic band structure properties of noncentrosymmetric Th7Fe3 superconductor.

Noncentrosymmetric superconductor Th7Fe3 has been investigated by means of specific heat, electrical resisitivity measurements and electronic properties calculations. Sudden drop in the resistivity at 2.05 ± 0.15 K and specific heat jump at 1.98 ± 0.02 K are observed, rendering the superconducting transition. A model of two BCS-type gaps appears to describe the zero-magnetic-field specific heat better than those based on the isotropic BCS theory or anisotropic functions. A positive curvature of the upper critical field H c2(T c) and nonlinear field dependence of the Sommerfeld coefficient at 0.4 K qualitatively support the two-gap scenario, which predicts H c2(0) = 13 kOe. The theoretical densities of states and electronic band structures (EBS) around the Fermi energy show a mixture of Th 6d- and Fe 3d-electrons bands, being responsible for the superconductivity. Furthermore, the EBS and Fermi surfaces disclose significantly anisotropic splitting associated with asymmetric spin-orbit coupling (ASOC). The ASOC sets up also multiband structure, which presumably favours a multigap superconductivity. Electron Localization Function reveals the existence of both metallic and covalent bonds, the latter may have different strengths depending on the regions close to the Fe or Th atoms. The superconducting, electronic properties and implications of asymmetric spin-orbit coupling associated with noncentrosymmetric structure are discussed.

Specific heat and Hall effect of the ferromagnetic Kondo lattice UCu0.9Sb2.

We have investigated the electrical resistivity ρ, specific heat C p and Hall coefficient R H on a single crystal of a ferromagnetic Kondo lattice UCu0.9Sb2. The experimental [Formula: see text], C p (T) and [Formula: see text] data evidence a bulk magnetic phase transition at [Formula: see text] K, and additionally exhibit an unexpected bump located in the temperature range T C/10-T C/3. UCu0.9Sb2 has an enhanced electronic specific heat coefficient [Formula: see text] mJ molK-2, corresponding to Kondo temperature [Formula: see text] K. An analysis of the Hall effect data for j//(a, b)-plane and H// c-axis reveals that the low-temperature ordinary Hall coefficient R 0 is positive, suggesting that p-type electrical conductivity is dominant. The density of the carriers at 2 K is about 0.6 holes f.u.-1, which may categorize the studied compound into class of low carrier density compounds. Combined γ and R 0 data divulge an effective mass of charge carriers [Formula: see text] 27 m e . This finding together with quite low Hall mobility [Formula: see text] cm2 Vs-1 and Kadowaki-Woods ratio [Formula: see text] [Formula: see text] cm (mol K2 mJ-1)2, manifest the development of heavy-fermion state in the ferromagnetic UCu0.9Sb2 compound at low temperatures.

How to improve breeding value prediction for feed conversion ratio in the case of incomplete longitudinal body weights.

With the development of automatic self-feeders, repeated measurements of feed intake are becoming easier in an increasing number of species. However, the corresponding BW are not always recorded, and these missing values complicate the longitudinal analysis of the feed conversion ratio (FCR). Our aim was to evaluate the impact of missing BW data on estimations of the genetic parameters of FCR and ways to improve the estimations. On the basis of the missing BW profile in French Large White pigs (male pigs weighed weekly, females and castrated males weighed monthly), we compared 2 different ways of predicting missing BW, 1 using a Gompertz model and 1 using a linear interpolation. For the first part of the study, we used 17,398 weekly records of BW and feed intake recorded over 16 consecutive weeks in 1,222 growing male pigs. We performed a simulation study on this data set to mimic missing BW values according to the pattern of weekly proportions of incomplete BW data in females and castrated males. The FCR was then computed for each week using observed data (obser_FCR), data with missing BW (miss_FCR), data with BW predicted using a Gompertz model (Gomp_FCR), and data with BW predicted by linear interpolation (interp_FCR). Heritability (h) was estimated, and the EBV was predicted for each repeated FCR using a random regression model. In the second part of the study, the full data set (males with their complete BW records, castrated males and females with missing BW) was analyzed using the same methods (miss_FCR, Gomp_FCR, and interp_FCR). Results of the simulation study showed that h were overestimated in the case of missing BW and that predicting BW using a linear interpolation provided a more accurate estimation of h and of EBV than a Gompertz model. Over 100 simulations, the correlation between obser_EBV and interp_EBV, Gomp_EBV, and miss_EBV was 0.93 ± 0.02, 0.91 ± 0.01, and 0.79 ± 0.04, respectively. The heritabilities obtained with the full data set were quite similar for miss_FCR, Gomp_FCR, and interp_FCR. In conclusion, when the proportion of missing BW is high, genetic parameters of FCR are not well estimated. In French Large White pigs, in the growing period extending from d 65 to 168, prediction of missing BW using a Gompertz growth model slightly improved the estimations, but the linear interpolation improved the estimation to a greater extent. This result is due to the linear rather than sigmoidal increase in BW over the study period.

Genetic structured antedependence and random regression models applied to the longitudinal feed conversion ratio in growing Large White pigs.

The objective of the present study was to compare a random regression model, usually used in genetic analyses of longitudinal data, with the structured antedependence (SAD) model to study the longitudinal feed conversion ratio (FCR) in growing Large White pigs and to propose criteria for animal selection when used for genetic evaluation. The study was based on data from 11,790 weekly FCR measures collected on 1,186 Large White male growing pigs. Random regression (RR) using orthogonal polynomial Legendre and SAD models was used to estimate genetic parameters and predict FCR-based EBV for each of the 10 wk of the test. The results demonstrated that the best SAD model (1 order of antedependence of degree 2 and a polynomial of degree 2 for the innovation variance for the genetic and permanent environmental effects, i.e., 12 parameters) provided a better fit for the data than RR with a quadratic function for the genetic and permanent environmental effects (13 parameters), with Bayesian information criteria values of -10,060 and -9,838, respectively. Heritabilities with the SAD model were higher than those of RR over the first 7 wk of the test. Genetic correlations between weeks were higher than 0.68 for short intervals between weeks and decreased to 0.08 for the SAD model and -0.39 for RR for the longest intervals. These differences in genetic parameters showed that, contrary to the RR approach, the SAD model does not suffer from border effect problems and can handle genetic correlations that tend to 0. Summarized breeding values were proposed for each approach as linear combinations of the individual weekly EBV weighted by the coefficients of the first or second eigenvector computed from the genetic covariance matrix of the additive genetic effects. These summarized breeding values isolated EBV trajectories over time, capturing either the average general value or the slope of the trajectory. Finally, applying the SAD model over a reduced period of time suggested that similar selection choices would result from the use of the records from the first 8 wk of the test. To conclude, the SAD model performed well for the genetic evaluation of longitudinal phenotypes.

Photoemission studies and electronic structure of U(2)T(2)In (T = Ni, Rh, Pt) compounds.

The electronic structure of the tetragonal U(2)T(2)In (T = Ni, Rh, Pt) compounds in the paramagnetic phase were studied by x-ray photoelectron spectroscopy (XPS). Both valence band and core level spectra were analysed. The experimental data are compared with the calculations of the density of states using the tight-binding linear muffin-tin orbital method (TB-LMTO) and full-potential local-orbital full-relativistic method (FPLO). The calculated data reveal a dominant U 5f electron character for the states near the Fermi level E(F) with a small contribution from U 5d, Ni 3d, Rh 4d, Pt 5d and In 5p states. The XPS valence bands of these compounds are characterized by a sharp peak of the U 5f states near the Fermi level (E(F)) and broad peaks of the Ni 3d, Rh 4d and Pt 5d states at about 2.6, 3.2 and 4.0 eV below E(F), respectively. The small change in the position of the U 5f peak with respect to E(F) is -0.35 eV for T = Ni and -0.15 eV for T = Rh and Pt. A satellite between the Ni 2p(1/2) and Ni 2p(3/2) peaks is visible, suggesting that the Ni 3d band is not completely filled, and the existence of a small induced magnetic moment on the Ni atoms cannot be ruled out.

Intermediate valence behaviour of Yb in a new intermetallic compound YbNi(0.8)Al(4.2).

We report on the valence band electronic structure as well as structural, magnetic and electronic transport properties of a novel intermetallic YbNi(0.8)Al(4.2). The compound crystallizes in the orthorhombic YNiAl(4)-type structure (a = 4.049(2),b = 15.305(5) and c = 6.586(3) Å) with space group Cmcm. The valence band spectrum and magnetic data indicate an intermediate valency of Yb ions in the studied compound with a valence of about 2.66 at room temperature. The temperature dependences of electrical resistivity, Hall effect and thermoelectric power display characteristic features of Kondo lattice with a large Kondo temperature (∼1300 K), being consistent with the intermediate valence scenario. An analysis of magnetic data suggests the presence of strong ferromagnetic correlations in the investigated compound at low temperatures. In order to obtain information about the electron-hole analogy between the Ce and Yb compounds, we have compared the observed behaviour with that of the dense Kondo compound CeNiAl(4).

Physical properties and electronic band structure of noncentrosymmetric Th7Co3 superconductor.

The physical properties of the noncentrosymmetric superconductor Th7Co3 have been investigated by means of ac-magnetic susceptibility, magnetization, specific heat, electrical resistivity, magnetoresistance and Hall effect measurements. From these data it is established that Th7Co3 is a dirty type-II superconductor with [Formula: see text] K, [Formula: see text] and moderate electron-phonon coupling [Formula: see text]. Some evidences for anisotropic superconducting gap are found, including e.g. reduced specific heat jump ([Formula: see text]) at T c, diminished superconducting energy gap ([Formula: see text]) as compared to the BCS values, power law field dependence of the Sommerfeld coefficient at 0.4 K ([Formula: see text]), and a concave curvature of the [Formula: see text] line. The magnitudes of the thermodynamic critical field and the energy gap are consistent with mean-squared anisotropy parameter [Formula: see text]. The electronic specific heat in the superconducting state is reasonably fitted to an oblate spheroidal gap model. Calculations of scalar relativistic and fully relativistic electronic band structures reveal considerable differences in the degenerate structure, resulting from asymmetric spin-orbit coupling (ASOC). A large splitting energy of spin-up spin-down bands at the Fermi level E F, [Formula: see text] meV is observed and a sizeable ratio [Formula: see text] could classify the studied compound into the class of noncentrosymmetric superconductors with strong ASOC. The noncentrosymmetry of the crystal structure and the atomic relativistic effects are both responsible for an importance of ASOC in Th7Co3. The calculated results for the density of states show a Van Hove singularity just below E F and dominant role of the 6d electrons of Th to the superconductivity.

The influence of temperature, pressure and Ag doping on the physical properties of TiO2 nanoceramics.

Undoped and Ag-doped TiO2 ceramics have been prepared at temperatures between 500-1000 °C and under pressures up to 8 GPa. Their crystal structures and physical properties were investigated by means of EDX, SEM, TEM, X-ray powder diffraction, and magnetization M, specific heat Cp and electrical resistance ρ measurements. It is found that the anatase-structured As-cast powder transforms into rutile and columbite-type at 500 °C and 5.5 GPa. The stabilization of the latter phase is fulfilled under a pressure of 8 GPa and at temperatures above 800 °C. On the basis of experimental results, we conclude that the physical properties of TiO2 can be tailored along with its crystal structure. In particular, magnetic properties change from paramagnetic in anatase and rutile to magnetic correlations and in all likelihood magnetic-field-induced antiferromagnetic short-range order in columbite-structured TiO2. Contrasting behaviour in the temperature dependences of specific heat between anatase/rutile and columbite-type TiO2 is obvious. Differently from anatase/rutile, the Cp of columbite-type TiO2 exhibits a low-temperature excess, being interpreted as due to magnetic correlations, or else the prevalence of soft modes. An analysis of ρ(T) for columbite-type TiO2 in the temperature range of 280-400 K reveals the presence of a new trapping state at an energy level of ∼28 meV within the originally forbidden gap. Furthermore, thermal fluctuation-induced tunnelling and hopping conductivities are suggested to govern in a lower temperature range. We recognize that the Ag-doped contents do not alter the crystal structure but considerably enhance magnetic correlations, compared to undoped samples.

Gingerols and related analogues inhibit arachidonic acid-induced human platelet serotonin release and aggregation.

Gingerols, the active components of ginger (the rhizome of Zingiber officinale, Roscoe), represent a potential new class of platelet activation inhibitors. In this study, we examined the ability of a series of synthetic gingerols and related phenylalkanol analogues (G1-G7) to inhibit human platelet activation, compared to aspirin, by measuring their effects on arachidonic acid (AA)-induced platelet serotonin release and aggregation in vitro. The IC(50) for inhibition of AA-induced (at EC(50)=0.75 mM) serotonin release by aspirin was 23.4+/-3.6 microM. Gingerols and related analogues (G1-G7) inhibited the AA-induced platelet release reaction in a similar dose range as aspirin, with IC(50) values between 45.3 and 82.6 microM. G1-G7 were also effective inhibitors of AA-induced human platelet aggregation. Maximum inhibitory (IC(max)) values of 10.5+/-3.9 and 10.4+/-3.2 microM for G3 and G4, respectively, were approximately 2-fold greater than aspirin (IC(max)=6.0+/-1.0 microM). The remaining gingerols and related analogues maximally inhibited AA-induced platelet aggregation at approximately 20-25 microM. The mechanism underlying inhibition of the AA-induced platelet release reaction and aggregation by G1-G7 may be via an effect on cyclooxygenase (COX) activity in platelets because representative gingerols and related analogues (G3-G6) potently inhibited COX activity in rat basophilic leukemia (RBL-2H3) cells. These results provide a basis for the design of more potent synthetic gingerol analogues, with similar potencies to aspirin, as platelet activation inhibitors with potential value in cardiovascular disease.

Performance measurements of a depth-encoding PET detector module based on position-sensitive avalanche photodiode read-out.

We are developing a high-resolution, high-efficiency positron emission tomography (PET) detector module with depth of interaction (DOI) capability based on a lutetium oxyorthosilicate (LSO) scintillator array coupled at both ends to position-sensitive avalanche photodiodes (PSAPDs). In this paper we present the DOI resolution, energy resolution and timing resolution results for complete detector modules. The detector module consists of a 7 x 7 matrix of LSO scintillator crystals (1 x 1 x 20 mm3 in dimension) coupled to 8 x 8 mm2 PSAPDs at both ends. Flood histograms were acquired and used to generate crystal look-up tables. The DOI resolution was measured for individual crystals within the array by using the ratio of the signal amplitudes from the two PSAPDs on an event-by-event basis. A measure of the total scintillation light produced was obtained by summing the signal amplitudes from the two PSAPDs. This summed signal was used to measure the energy resolution. The DOI resolution was measured to be 3-4 mm FWHM irrespective of the position of the crystal within the array, or the interaction location along the length of the crystal. The total light signal and energy resolution was almost independent of the depth of interaction. The measured energy resolution averaged 14% FWHM. The coincidence timing resolution measured using a pair of identical detector modules was 4.5 ns FWHM. These results are consistent with the design goals and the performance required of a compact, high-resolution and high-efficiency PET detector module for small animal and breast imaging applications.

The stability of gingerol and shogaol in aqueous solutions.

Gingerols, pungent principles of ginger (the rhizome of Zingiber officinale), are biologically active components that may make a significant contribution towards medicinal applications of ginger and some products derived from ginger. Gingerols, however, are thermally labile due to the presence of a beta-hydroxy keto group in the structure, and undergo dehydration readily to form the corresponding shogaols. This study investigated the stability of [6]-gingerol [5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decan-3-one] at temperatures ranging from 37 to 100 degrees C in aqueous solutions, at pH 1, 4, and 7. Quantitative measurements of [6]-gingerol and its major degradation product [6]-shogaol [1-(4-hydroxy-3-methoxyphenyl)decan-4-ene-3-one] were performed by HPLC. Kinetics of [6]-gingerol degradation was characterized by least square fitting of a rate equation. It was found that gingerol exhibited novel reversible kinetics, in which it undergoes dehydration-hydration transformations with shogaol, the major degradation product. Degradation rates were found to be pH dependent with greatest stability observed at pH 4. The reversible degradation of [6]-gingerol at 100 degrees C and pH 1 was relatively fast and reached equilibrium within 2 h. Activation energies for the forward and reverse reactions for [6]-gingerol were calculated from the Arrhenius equation using reaction rates obtained at temperatures ranging from 37 to 100 degrees C.

Fascioliasis in Vietnam.

A confirmed diagnosis of human fascioliasis was rare in Vietnam until 1978 when two cases were reported in humans. Since 1997, we have confirmed 500 cases of human fascioliasis. The majority of cases come from the central provinces of Vietnam: Da Nang, Quang Ngai, Binh Dinh, Phu Yen and Khanh Hoa. Patients were treated in hospitals in Ho Chi Minh City. All had high peripheral blood eosinophilic counts (16-70%) and positive serology with Fasciola gigantica antigen with positive titers of 1/1,600 to 1/12,800. We are unsure whether this represents an endemic pattern of disease or whether improved specific laboratory tools now facilitate better diagnosis. It is also possible that with changes in environmental factors and in the number and breeds of herbivorous domestic animals, Fasciola is increasing in frequency and easily contaminates the food.

Bak apoptotic function is not directly regulated by phosphorylation.

During apoptosis, Bak and Bax permeabilize the mitochondrial outer membrane by undergoing major conformational change and oligomerization. This activation process in Bak is reported to require dephosphorylation of tyrosine-108 close to an activation trigger site. To investigate how dephosphorylation of Bak contributes to its activation and conformational change, one-dimensional isoelectric focusing (1D-IEF) and mutagenesis was used to monitor Bak phosphorylation. On 1D-IEF, Bak extracted from a range of cell types migrated as a single band near the predicted isoelectric point of 5.6 both before and after phosphatase treatment, indicating that Bak is not significantly phosphorylated at any residue. In contrast, three engineered 'phosphotagged' Bak variants showed a second band at lower pI, indicating phosphorylation. Apoptosis induced by several stimuli failed to alter Bak pI, indicating little change in phosphorylation status. In addition, alanine substitution of tyrosine-108 and other putative phosphorylation sites failed to enhance Bak activation or pro-apoptotic function. In summary, Bak is not significantly phosphorylated at any residue, and Bak activation during apoptosis does not require dephosphorylation.