A cluster pattern algorithm for the analysis of multiparametric cell assays.

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.     

Human p38α mitogen-activated protein kinase in the Asp168-Phe169-Gly170-in (DFG-in) state can bind allosteric inhibitor Doramapimod

Doramapimod (BIRB-796) is widely recognized as one of the most potent and selective type II inhibitors of human p38α mitogen-activated protein kinase (MAPK); however, the understanding of its binding mechanism remains incomplete. Previous studies indicated high affinity of the ligand to a so-called allosteric pocket revealed only in the ‘out’ state of the DFG motif (i.e. Asp168-Phe169-Gly170) when Phe169 becomes fully exposed to the solvent. The possibility of alternative binding in the DFG-in state was hypothesized, but the molecular mechanism was not known. Methods of bioinformatics, docking and long-time scale classical and accelerated molecular dynamics have been applied to study the interaction of Doramapimod with the human p38α MAPK. It was shown that Doramapimod can bind to the protein even when the Phe169 is fully buried inside the allosteric pocket and the kinase activation loop is in the DFG-in state. Orientation of the inhibitor in such a complex is significantly different from that in the known crystallographic complex formed by the kinase in the DFG-out state; however, the Doramapimod’s binding is followed by the ligand-induced conformational changes, which finally improve accommodation of the inhibitor. Molecular modelling has confirmed that Doramapimod combines the features of type I and II inhibitors of p38α MAPK, i.e. can directly and indirectly compete with the ATP binding. It can be concluded that optimization of the initial binding in the DFG-in state and the final accommodation in the DFG-out state should be both considered at designing novel efficient type II inhibitors of MAPK and homologous proteins.

Communicated by Ramaswamy H. Sarma     

Blood–Brain Barrier Breakdown in Stress and Neurodegeneration: Biochemical Mechanisms and New Models for Translational Research

Biochemistry (Moscow) - Blood-brain barrier (BBB) is a structural and functional element of the neurovascular unit (NVU), which includes cells of neuronal, glial, and endothelial nature. The main...     

I. Primexine development in Passiflora racemosa Brot.: overlooked aspects of development

Developmental stages during the tetrad period were examined in detail by transmission electron microscopy with an emphasis on substructure. Our purpose was to find out whether the sequence of sporoderm developmental events provides additional evidence for our recent hypothesis on the underlying cause of exine ontogeny as a sequence of self-assembling micellar mesophases initiated by genomically given physicochemical parameters. Osmiophilic globules encrusting the surface of postmeiotic microspores and tapetal cells are temporary prepattern units which come first. The second prepattern structures are highly ordered bundles of microfilaments and microtubules which determine the position of microspore surface invaginations and clusters of the glycocalyx inside them. The first glycocalyx units are microgranules which during the middle tetrad stage rearrange into radially oriented rod-like units. The latter form lens-like clusters of the glycocalyx-1, located inside the invaginations. These clusters predestine the position of the future luminae in the exine reticulum. The second glycocalyx layer is laid down as a continuous layer over the whole microspore surface and has similar substructure, that is radial rods. Glycocalyx-2 is a framework for procolumellae which appear at the late tetrad stage. Therefore, the sequence of substructural units in the primexine is: globules, microgranules, rod-like units, and layers of radially oriented rods tightly packed in the periplasmic space. This sequence corresponds to the first three mesophases of self-assembling micelles: spherical micelles, cylindrical micelles, and layers of hexagonally packed cylindrical micelles (middle mesophase). We observed the same sequence in other species during primexine development, and the findings of this study provide new evidence for our hypothesis.     

The Barnes Maze Task Reveals Specific Impairment of Spatial Learning Strategy in the Intrahippocampal Kainic Acid Model for Temporal Lobe Epilepsy

Neurochemical Research - Temporal lobe epilepsy (TLE) is an acquired form of focal epilepsy, in which patients not only suffer from unprovoked, devastating seizures, but also from severe...     

Stress reactions of Scots pine trees to injuring by ground fire

Controlled burning has been carried out to stimulate moderate ground fire in the Scots pine (Pinus sуlvestris L.) stand (age class I) in the Krasnoyarsk forest steppe at the beginning of the growing season. The structure of annual rings and trophic supply, as well as the cambial zone antioxidant system condition and the content of photosynthetic pigments in the needles of dominant and suppressed trees in the experimental and control plots, were studied 3 h, 5 days, and 2.5 months after the fire. The fire narrowed the annual ring width, since the number and size of early and late tracheids decreased. The primary reaction to pyrogenic heat shock (sharp reduction of chlorophyll content in needles and manifestation of oxidative stress in the stem cambial zone) was an elevated level of hydrogen peroxide and activity of most antioxidant enzymes and a reduction in the nonstructural carbohydrates pool. Restoration of the cambium function after the fire was promoted by an increased activity of amylase, invertase, and peroxidase.     

Effects of particulate matter exposure on blood 5-hydroxymethylation: results from the Beijing truck driver air pollution study

Previous studies have reported epigenetic changes induced by environmental exposures. However, previous investigations did not distinguish 5-methylcytosine (5mC) from a similar oxidative form with opposite functions, 5-hydroxymethylcytosine (5hmC). Here, we measured blood DNA global 5mC and 5hmC by ELISA and used adjusted mixed-effects regression models to evaluate the effects of ambient PM₁₀ and personal PM_2.5 and its elemental components—black carbon (BC), aluminum (Al), calcium (Ca), potassium (K), iron (Fe), sulfur (S), silicon (Si), titanium (Ti), and zinc (Zn)—on blood global 5mC and 5hmC levels. The study was conducted in 60 truck drivers and 60 office workers in Beijing, China from The Beijing Truck Driver Air Pollution Study at 2 exams separated by one to 2 weeks. Blood 5hmC level (0.08%) was ∼83-fold lower than 5mC (6.61%). An inter-quartile range (IQR) increase in same-day PM₁₀ was associated with increases in 5hmC of 26.1% in office workers (P = 0.004), 20.2% in truck drivers (P = 0.014), and 21.9% in all participants combined (P < 0.001). PM₁₀ effects on 5hmC were increasingly stronger when averaged over 4, 7, and 14 d preceding assessment (up to 132.6% for the 14-d average in all participants, P < 0.001). PM₁₀ effects were also significant after controlling for multiple testing (family-wise error rate; FWER < 0.05). 5hmC was not correlated with personal measures of PM_2.5 and elemental components (FWER > 0.05). 5mC showed no correlations with PM₁₀, PM_2.5, and elemental components measures (FWER > 0.05). Our study suggests that exposure to ambient PM₁₀ affects 5hmC over time, but not 5mC. This finding demonstrates the need to differentiate 5hmC and 5mC in environmental studies of DNA methylation.     

The role of phosphate groups in the VS ribozyme-substrate interaction

The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme–substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem–loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.