Structural mechanism of transcription regulation of the Staphylococcus aureus multidrug efflux operon mepRA by the MarR family repressor MepR

The multidrug efflux pump MepA is a major contributor to multidrug resistance in Staphylococcus aureus. MepR, a member of the multiple antibiotic resistance regulator (MarR) family, represses mepA and its own gene. Here, we report the structure of a MepR–mepR operator complex. Structural comparison of DNA-bound MepR with ‘induced’ apoMepR reveals the large conformational changes needed to allow the DNA-binding winged helix-turn-helix motifs to interact with the consecutive major and minor grooves of the GTTAG signature sequence. Intriguingly, MepR makes no hydrogen bonds to major groove nucleobases. Rather, recognition-helix residues Thr60, Gly61, Pro62 and Thr63 make sequence-specifying van der Waals contacts with the TTAG bases. Removing these contacts dramatically affects MepR–DNA binding activity. The wings insert into the flanking minor grooves, whereby residue Arg87, buttressed by Asp85, interacts with the O2 of T₄ and O4′ ribosyl oxygens of A₂₃ and T₄. Mutating Asp85 and Arg87, both conserved throughout the MarR family, markedly affects MepR repressor activity. The His14′:Arg59 and Arg10′:His35:Phe108 interaction networks stabilize the DNA-binding conformation of MepR thereby contributing significantly to its high affinity binding. A structure-guided model of the MepR–mepA operator complex suggests that MepR dimers do not interact directly and cooperative binding is likely achieved by DNA-mediated allosteric effects.     

Mutation Analysis of the ERCC4/FANCQ Gene in Hereditary Breast Cancer

The ERCC4 protein forms a structure-specific endonuclease involved in the DNA damage response. Different cancer syndromes such as a subtype of Xeroderma pigmentosum, XPF, and recently a subtype of Fanconi Anemia, FA-Q, have been attributed to biallelic ERCC4 gene mutations. To investigate whether monoallelic ERCC4 gene defects play some role in the inherited component of breast cancer susceptibility, we sequenced the whole ERCC4 coding region and flanking untranslated portions in a series of 101 Byelorussian and German breast cancer patients selected for familial disease (set 1, n = 63) or for the presence of the rs1800067 risk haplotype (set 2, n = 38). This study confirmed six known and one novel exonic variants, including four missense substitutions but no truncating mutation. Missense substitution p.R415Q (rs1800067), a previously postulated breast cancer susceptibility allele, was subsequently screened for in a total of 3,698 breast cancer cases and 2,868 controls from Germany, Belarus or Russia. The Gln415 allele appeared protective against breast cancer in the German series, with the strongest effect for ductal histology (OR 0.67; 95%CI 0.49; 0.92; p = 0.003), but this association was not confirmed in the other two series, with the combined analysis yielding an overall Mantel-Haenszel OR of 0.94 (95% CI 0.81; 1.08). There was no significant effect of p.R415Q on breast cancer survival in the German patient series. The other three detected ERCC4 missense mutations included two known rare variants as well as a novel substitution, p.E17V, that we identified on a p.R415Q haplotype background. The p.E17V mutation is predicted to be probably damaging but was present in just one heterozygous patient. We conclude that the contribution of ERCC4/FANCQ coding mutations to hereditary breast cancer in Central and Eastern Europe is likely to be small.     

Reproducibility of Functional Connectivity and Graph Measures Based on the Phase Lag Index (PLI) and Weighted Phase Lag Index (wPLI) Derived from High Resolution EEG

Functional connectivity (FC) and graph measures provide powerful means to analyze complex networks. The current study determines the inter-subject-variability using the coefficient of variation (CoV) and long-term test-retest-reliability (TRT) using the intra-class correlation coefficient (ICC) in 44 healthy subjects with 35 having a follow-up at years 1 and 2. FC was estimated from 256-channel-EEG by the phase-lag-index (PLI) and weighted PLI (wPLI) during an eyes-closed resting state condition. PLI quantifies the asymmetry of the distribution of instantaneous phase differences of two time-series and signifies, whether a consistent non-zero phase lag exists. WPLI extends the PLI by additionally accounting for the magnitude of the phase difference. Signal-space global and regional PLI/wPLI and weighted first-order graph measures, i.e. normalized clustering coefficient (gamma), normalized average path length (lambda), and the small-world-index (SWI) were calculated for theta-, alpha1-, alpha2- and beta-frequency bands. Inter-subject variability of global PLI was low to moderate over frequency bands (0.12<CoV<0.28), higher for wPLI (0.25<CoV<0.55) and very low for gamma, lambda and SWI (CoV<0.048). TRT was good to excellent for global PLI/wPLI (0.68<ICC<0.80), regional PLI/wPLI (0.58<ICC<0.77), and fair to good for graph measures (0.32<ICC<0.73) except wPLI-based lambda in alpha1 (ICC = 0.12). Inter-electrode distance correlated very weakly with inter-electrode PLI (−0.06<rho<0) and weakly with inter-electrode wPLI (−0.22<rho<−0.18). Global PLI/wPLI and topographic connectivity patterns differed between frequency bands, and all individual networks showed a small-world-configuration. PLI/wPLI based network characterization derived from high-resolution EEG has apparently good reliability, which is one important requirement for longitudinal studies exploring the effects of chronic brain diseases over several years.     

Effects of aquatic insect predators on zooplankton in fishless ponds

We removed the surface-orienting aquatic insects from a fishless pond to determine their predation effects on zooplankton behavior and size structure. A second fishless pond served as the unmanipulated reference system in this two year study. In the reference pond and the treatment pond prior to manipulation, daphnids exhibited pronounced diel vertical migrations. Following the removal of surface-orienting aquatic insects from the treatment pond, daphnid migration changed to a reverse migration strategy that was significantly different from that observed in the reference system. Average daphnid body size increased significantly following predator removal in the treatment system. Our data indicate that predation by aquatic insect predators, such as notonectids and dytiscids, may affect daphnid migration behavior in fishless systems. Vertical migration by daphnids may allow coexistence with surface-orienting insects in ponds that are deep enough to provide a spatial refuge from these predators.     

Flow cytometry as a new method for the measurement of electrophoretic mobility of erythrocytes using membrane charge staining by fluoresceinated polycations.

The binding of FITC-labeled poly-L-ornithine and poly-L-lysine to fresh or neuraminidase treated human, rat or rabbit erythrocytes was investigated by simultaneous cell volume and cell membrane fluorescence measurements in a flow cytometer. The cell volume was converted into cell surface and the distribution curve of the fluorescence/micrometer2 cell surface was calculated from all histogram classes by a computer program. The mean fluorescence/micrometer2 cell surface as a measure of the density of the negative charges on the cell surface was directly proportional to the elctrophoretic mobility of the erythrocytes, showing that polycation binding can effectively be used for the measurement of the electrophoretic mobility of erythrocytes. The computer fitting of the experimental two parameter histograms by two dimensional Gaussian normal distributions was found to be a very efficient way of data reduction, and a good separation of overlapping cell clusters was possible even in the case of low total numbers of cells in the histogram.     

Metabolism and motility of turkey (Meleagris gallopavo) spermatozoa in the presence or absence of oxygen, glucose and fructose

Sugar consumption by turkey spermatozoa for 1 hr at 37 degrees C was similar in the presence or absence of oxygen, and with glucose or fructose in the medium. Motility of the spermatozoa at the end of the above incubation period was lower under anaerobic than aerobic conditions. Fructose enhanced the oxygen uptake of spermatozoa in comparison with that in the glucose-containing medium. Omission of sugar from the medium depressed respiration but not motility of the spermatozoa. The rate of oxygen uptake by the spermatozoa during a 3-hr incubation period was higher in a 3.3-mM than a 15.0-mM glucose medium. Fructose was formed from glucose under aerobic but not under anaerobic conditions. Fructose originating from glucose was used for fructolysis, when the glucose reserve in the medium was almost exhausted.     

The effect of mating,artificial insemination,and fright on uterine motility of the oestrous ewe

Pressure changes inside the uterine wall were recorded by telemetry in six oestrous Awassi ewes during natural hand mating and AI, and in two oestrous ewes during fright caused by the presence of dogs.During control periods, the average frequency of primary pressure peaks was 3.7/min, their average amplitude was 29.6 cm H₂O pressure, and there was an average of 2.5 secondary peaks per primary pressure peak. During AI or natural mating these parameters were 1.1–1.7 and during fright 1.1–1.3 times their control values, on the average. The experimental treatments caused practically no after effects.The results do not support the hypothesis that sperm transport in artificially inseminated ewes is impaired by inhibition of uterine motility. Other explanations are offered.