Altered proteome profiles in maternal plasma in pregnancies with fetal growth restriction

Fetal growth restriction (FGR) affects 3–5% of pregnancies and is associated with increased perinatal morbidity and mortality. Currently, there is no reliable biochemical test to differentiate a pathological FGR from a nonpathological one. The objective of this study was to screen whole maternal plasma to identify differentially expressed relatively abundant proteins associated with FGR. We analyzed maternal plasma from FGR (n=28) and healthy (n=22) pregnancies using two-dimensional gel electrophoresis (2D-GE) followed by software image analysis. Three spots with molecular weight (M_r) 18 kDa corresponding to haptoglobin (hp) α2, as identified by LC-MS/MS and immunoblotting, showed differential expression patterns in FGR. The distribution of hp α2 variants in maternal plasma samples showed the hp α2 variant 1 was low in 72% of FGR, medium in 16%, whereas high in 12%. In comparison, hp α2 variant 1 was high in (41%) of controls, medium in 41%, and low in 18% of cases. Based on the software image analysis, the mean spot volume for hp α2 variant 1 was 0.12 (SD=0.18) for FGR compared to 0.26 (SD=0.19) for control (p=0.006). Given that hp turnover is indicative of its maturation process and is traceable in plasma by its dominant/suppressed variants, we propose that hp α2 is an important potential target for evaluation of its clinical and pathophysiological role and as a diagnostic biomarker in FGR.     

Quantitative 2-D gel electrophoresis-based expression proteomics of albumin and IgG immunodepleted plasma

Proteomic analysis of plasma is challenging because of its large dynamic range, which prevents the detection of low abundance proteins. Immunodepletion of high abundance proteins, such as albumin and IgG, has emerged as a favored technology to overcome this problem; however its suitability in quantitative expression proteomics has not yet been adequately addressed. In this study, albumin and IgG immunodepletion was evaluated by ELISAs and the reproducibility of depletion was tested with 2-DGE. Depletion of plasma resulted in removal of 62+/-1.2% of the total protein, 93+/-1.4% of the albumin (0.43 microg/microL, residual), and 94+/-1.5% of the IgG (0.21 microg/microL, residual). These results were confirmed by immunoblotting. Computerized image analysis of 2-D gels using Progenesis SameSpots software revealed an enhancement in the number of visible spots (675-1325), with 10+/-6% inter-gel variability in spot density. LC-ESI-MS/MS identification of newly resolved protein spots further validated the procedure. An innovative application of the software employed led to identification of 11 proteins lost non-specifically during depletion. This study demonstrates the effectiveness of immunodepletion of albumin and IgG in quantitative 2-DGE-based differential analysis of plasma proteins.     

Biophysical modeling of in vitro and in vivo processes underlying regulated photoprotective mechanism in cyanobacteria

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10^?5 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

     

Two Radix spp. (Gastropoda: Lymnaeidae) endemic to thermal springs around Lake Baikal represent ecotypes of the widespread Radix auricularia

In this study, we re‐examine two species of freshwater gastropods of the genus Radix Montfort, 1810 (family Lymnaeidae), endemic to the geothermal springs in the Lake Baikal region in the southern part of eastern Siberia — Lymnaea (Radix) hakusyensis Kruglov et Starobogatov, 1989, and Lymnaea (Radix) thermobaicalica Kruglov et Starobogatov, 1989. The alleged species status of these endemics has been re‐assessed by means of an integrative approach combining molecular genetic taxonomy techniques with the traditional methods based on shell and soft body morphology. Phylogenetic reconstructions were made using both mitochondrial (COI) and nuclear (ITS2) DNA markers. We used topotypic samples of both species and specimens sampled from other sites around Lake Baikal. The results demonstrate that the two endemic species are only synonyms of a widespread Holarctic species, Radix auricularia (Linnaeus, 1758), and represent its intraspecific morph (ecotype) adapted to living in thermal springs. A new synonymy is proposed: Thermoradix Kruglov et Starobogatov, 1989 = Radix Montfort, 1810 (syn. n.).     

Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.     

Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells.

BACKGROUND: Chemical cytometry is an emerging technology that analyzes chemical contents of single cells by means of capillary electrophoresis or capillary chromatography. It has a potential to become an indispensable tool in analyses of heterogeneous cell populations such as those in tumors. Ras oncogenes are found in 30% of human cancers. To become fully functional products, oncogenic Ras proteins require at least three posttranslational modifications: farnesylation, endoproteolysis, and carboxyl-methylation. Therefore, enzymes that catalyze the three reactions, farnesyltransferase (FTase), endoprotease (EPase), and methyltransferase (MTase), are considered highly attractive therapeutic targets. In this work, we used chemical cytometry to study the metabolism of a pentapeptide substrate that can mimic Ras proteins with respect to their posttranslational modifications in solution. METHODS: Mouse mammary gland tumor cells (4T1) and mouse embryo fibroblasts (NIH3T3) were incubated with a fluorescently labeled pentapeptide substrate, 2',7'-difluorofluorescein-5-carboxyl-Gly-Cys-Val-Ilu-Ala. Cells were washed from the substrate and resuspended in phosphate buffered saline. Uptake of the substrate by the cells was monitored by laser scanning confocal microscopy. Single cells were injected into the capillary, lysed, and subjected to capillary electrophoresis. Fluorescent metabolic products were detected by laser-induced fluorescence and compared with products obtained by the conversion of the substrate by FTase, EPase, and MTase in solution. Co-sampling of single cells with the in-vitro products was used for such comparison. RESULTS: Confocal microscopy data showed that the substrate permeated the plasma membrane and clustered in the cytoplasm. Further capillary electrophoresis and chemical cytometry analyses showed that the substrate was converted into three fluorescently labeled products, two of which were secreted in the culture medium and one remained in the cells. The intracellular product was present at approximately 100,000 molecules per cell. The three metabolic products of the substrate were found to be different from the products of its processing by FTase, EPase, and MTase in solution. CONCLUSIONS: This is the first report of chemical cytometry in the context of Ras-signaling studies. The chemical cytometry method used in this work will find applications in the development of suitable peptide substrates for monitoring enzyme activities in single cells.     

Dynamics of the exponential integrate-and-fire model with slow currents and adaptation

In order to properly capture spike-frequency adaptation with a simplified point-neuron model, we study approximations of Hodgkin-Huxley (HH) models including slow currents by exponential integrate-and-fire (EIF) models that incorporate the same types of currents. We optimize the parameters of the EIF models under the external drive consisting of AMPA-type conductance pulses using the current-voltage curves and the van Rossum metric to best capture the subthreshold membrane potential, firing rate, and jump size of the slow current at the neuron’s spike times. Our numerical simulations demonstrate that, in addition to these quantities, the approximate EIF-type models faithfully reproduce bifurcation properties of the HH neurons with slow currents, which include spike-frequency adaptation, phase-response curves, critical exponents at the transition between a finite and infinite number of spikes with increasing constant external drive, and bifurcation diagrams of interspike intervals in time-periodically forced models. Dynamics of networks of HH neurons with slow currents can also be approximated by corresponding EIF-type networks, with the approximation being at least statistically accurate over a broad range of Poisson rates of the external drive. For the form of external drive resembling realistic, AMPA-like synaptic conductance response to incoming action potentials, the EIF model affords great savings of computation time as compared with the corresponding HH-type model. Our work shows that the EIF model with additional slow currents is well suited for use in large-scale, point-neuron models in which spike-frequency adaptation is important.     

Age-Related Loss of Lumbar Spinal Lordosis and Mobility – A Study of 323 Asymptomatic Volunteers

Background

The understanding of the individual shape and mobility of the lumbar spine are key factors for the prevention and treatment of low back pain. The influence of age and sex on the total lumbar lordosis and the range of motion as well as on different lumbar sub-regions (lower, middle and upper lordosis) in asymptomatic subjects still merits discussion, since it is essential for patient-specific treatment and evidence-based distinction between painful degenerative pathologies and asymptomatic aging.

Methods and Findings

A novel non-invasive measuring system was used to assess the total and local lumbar shape and its mobility of 323 asymptomatic volunteers (age: 20–75 yrs; BMI <26.0 kg/m²; males/females: 139/184). The lumbar lordosis for standing and the range of motion for maximal upper body flexion (RoF) and extension (RoE) were determined. The total lordosis was significantly reduced by approximately 20%, the RoF by 12% and the RoE by 31% in the oldest (>50 yrs) compared to the youngest age cohort (20–29 yrs). Locally, these decreases mostly occurred in the middle part of the lordosis and less towards the lumbo-sacral and thoraco-lumbar transitions. The sex only affected the RoE.

Conclusions

During aging, the lower lumbar spine retains its lordosis and mobility, whereas the middle part flattens and becomes less mobile. These findings lay the ground for a better understanding of the incidence of level- and age-dependent spinal disorders, and may have important implications for the clinical long-term success of different surgical interventions.     

Non-stressful death of 23S rRNA mutant G2061C defective in puromycin reaction

Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown. Here we report a study of the lethal G2061C mutant of Escherichia coli 23S ribosomal RNA (rRNA). The G2061C mutation completely impaired the puromycin reaction and abolished formation of the active firefly luciferase in an in vitro translation system, while poly(U)- and short synthetic mRNA-directed peptidyl transferase reaction with aminoacylated tRNAs in vitro was seemingly unaffected. Study of the cellular proteome upon expression of the 23S rRNA gene carrying the G2061C mutation compared to cells expressing wild-type 23S rRNA gene revealed substantial differences. Most of the observed effects in the mutant were associated with reduced expression of stress response proteins and particularly proteins associated with the ppGpp-mediated stringent response.