Fully automated counting of DNA damage foci in tumor cell culture: A matter of cell separation.

Analysis and quantification of residual, unrepaired DNA double-strand breaks by detecting damage-associated γH2AX or 53BP1 foci is a promising approach to evaluate radiosensitivity or radiosensitization in tumor cells. Manual foci quantification by eye is well-established but unsatisfactory due to inconsistent foci numbers between different observers, lack of information about foci size and intensity and the time-consuming scoring process. Therefore, automated foci counting is an important goal. Several software solutions for automated foci counting in separately acquired fluorescence microscopy images have been established. The AKLIDES NUK technology by Medipan combines automated microscopy and image processing/ counting, enabling affordable high throughput foci analysis as a routine application. Using this machine, automated foci counting is well established for lymphocytes but has not yet been reported for adherent tumor cells with their irregularly shaped nuclei and heterogeneous foci textures. Here we aimed to use the AKLIDES NUK system for adherent tumor cells growing in clusters. We identified cell separation as a critical step to ensure fast and reliable automated nuclei detection. We validated our protocol for the fully automated quantification of (i) the IR-dose dependent increase and (ii) the ATM as well as PARP inhibitor-induced radiosensitization. Collectively, with this protocol the AKLIDES NUK system facilitates cost effective, fast and high throughput quantitative fluorescence microscopic analysis of DNA damage induced foci such as γH2AX and 53BP1 in adherent tumor cells.

Chemical shift reference scale for Li solid state NMR derived by first-principles DFT calculations.

For studying electrode and electrolyte materials for lithium ion batteries, solid-state (SS) nuclear magnetic resonance (NMR) of lithium moves into focus of current research. Theoretical simulations of magnetic resonance parameters facilitate the analysis and interpretation of experimental Li SS-NMR spectra and provide unique insight into physical and chemical processes that are determining the spectral profile. In the present paper, the accuracy and reliability of the theoretical simulation methods of Li chemical shielding values is benchmarked by establishing a reference scale for Li SS-NMR of diamagnetic compounds. The impact of geometry, ionic mobility and relativity are discussed. Eventually, the simulation methods are applied to the more complex lithium titanate spinel (Li4Ti5O12, LTO), which is a widely discussed battery anode material. Simulation of the Li SS-NMR spectrum shows that the commonly adopted approach of assigning the resonances to individual crystallographic sites is not unambiguous.

Time-optimal excitation of maximum quantum coherence: Physical limits and pulse sequences.

Here we study the optimum efficiency of the excitation of maximum quantum (MaxQ) coherence using analytical and numerical methods based on optimal control theory. The theoretical limit of the achievable MaxQ amplitude and the minimum time to achieve this limit are explored for a set of model systems consisting of up to five coupled spins. In addition to arbitrary pulse shapes, two simple pulse sequence families of practical interest are considered in the optimizations. Compared to conventional approaches, substantial gains were found both in terms of the achieved MaxQ amplitude and in pulse sequence durations. For a model system, theoretically predicted gains of a factor of three compared to the conventional pulse sequence were experimentally demonstrated. Motivated by the numerical results, also two novel analytical transfer schemes were found: Compared to conventional approaches based on non-selective pulses and delays, double-quantum coherence in two-spin systems can be created twice as fast using isotropic mixing and hard spin-selective pulses. Also it is proved that in a chain of three weakly coupled spins with the same coupling constants, triple-quantum coherence can be created in a time-optimal fashion using so-called geodesic pulses.

Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation.

Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3'-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.

Visualization and analysis of modulated pulses in magnetic resonance by joint time-frequency representations.

We study the utility of joint time-frequency representations for the analysis of shaped or composite pulses for magnetic resonance. Such spectrograms are commonly used for the visualization of shaped laser pulses in optical spectroscopy. This intuitive representation provides additional insight compared to conventional approaches, which exclusively show either temporal or spectral information. We focus on the short-time Fourier transform, which provides not only amplitude but also phase information. The approach is illustrated for broadband inversion pulses, multiple quantum excitation and broadband heteronuclear decoupling. The physical interpretation and validity of the approach is discussed.

p53 modulates homologous recombination at I-SceI-induced double-strand breaks through cell-cycle regulation.

Inhibition of homologous recombination (HR) is believed to be a transactivation-independent function of p53 that protects from genetic instability. Misrepair by HR can lead to genetic alterations such as translocations, duplications, insertions and loss of heterozygosity, which all bear the risk of driving oncogenic transformation. Regulation of HR by wild-type p53 (wtp53) should prevent these genomic rearrangements. Mutation of p53 is a frequent event during carcinogenesis. In particular, dominant-negative mutants inhibiting wtp53 expressed from the unperturbed allel can drive oncogenic transformation by disrupting the p53-dependent anticancer barrier. Here, we asked whether the hot spot mutants R175H and R273H relax HR control in p53-proficient cells. Utilizing an I-SceI-based reporter assay, we observed a moderate (1.5 × ) stimulation of HR upon expression of the mutant proteins in p53-proficient CV-1, but not in p53-deficient H1299 cells. Importantly, the stimulatory effect was exactly paralleled by an increase in the number of HR competent S- and G2-phase cells, which can well explain the enhanced recombination frequencies. Furthermore, the impact on HR exerted by the transactivation domain double-mutant L22Q/W23S and mutant R273P, both of which were reported to regulate HR independently of G1-arrest execution, is also exactly mirrored by cell-cycle behavior. These results are in contrast to previous concepts stating that the transactivation-independent impact of p53 on HR is a general phenomenon valid for replication-associated and also for directly induced double-strand break. Our data strongly suggest that the latter is largely mediated by cell-cycle regulation, a classical transactivation-dependent function of p53.

[Inhibitor development after changing FVIII/IX products in patients with haemophilia]. / Hemmkörperentwicklung bei Hämophilie-Patienten nach Präparatewechsel. Steigt das Risiko beim Präparatewechsel an?

UNLABELLED: The retrospective observational study surveys the relationship between development of inhibitors in the treatment of haemophilia patients and risk factors such as changing FVIII products. A total of 119 patients were included in this study, 198 changes of FVIII products were evaluated. RESULTS: During the observation period of 12 months none of the patients developed an inhibitor, which was temporally associated with a change of FVIII products. A frequent change of FVIII products didn't lead to an increase in inhibitor risk. The change between plasmatic and recombinant preparations could not be confirmed as a risk factor. Furthermore, no correlation between treatment regimens, severity, patient age and comorbidities of the patients could be found.

Surface imaging using holographic optical tweezers.

We present an imaging technique using an optically trapped cigar-shaped probe controlled using holographic optical tweezers. The probe is raster scanned over a surface, allowing an image to be taken in a manner analogous to scanning probe microscopy (SPM), with automatic closed loop feedback control provided by analysis of the probe position recorded using a high speed CMOS camera. The probe is held using two optical traps centred at least 10 µm from the ends, minimizing laser illumination of the tip, so reducing the chance of optical damage to delicate samples. The technique imparts less force on samples than contact SPM techniques, and allows highly curved and strongly scattering samples to be imaged, which present difficulties for imaging using photonic force microscopy. To calibrate our technique, we first image a known sample--the interface between two 8 µm polystyrene beads. We then demonstrate the advantages of this technique by imaging the surface of the soft alga Pseudopediastrum. The scattering force of our laser applied directly onto this sample is enough to remove it from the surface, but we can use our technique to image the algal surface with minimal disruption while it is alive, not adhered and in physiological conditions. The resolution is currently equivalent to confocal microscopy, but as our technique is not diffraction limited, there is scope for significant improvement by reducing the tip diameter and limiting the thermal motion of the probe.

Linezolid treatment of nosocomial bacterial infection with multiresistant Gram-positive pathogens in preterm infants: a systematic review.

Linezolid is an antibiotic of the oxazolidinone class that has bacteriostatic and bactericidal activity against a broad range of Gram-positive bacteria, including multiresistant pathogens. Owing to increasing resistance of Gram-positive pathogens to traditional antibiotics such as vancomycin, the oxazolidinones were introduced into therapy. The aim of this review was to summarise actual data on the pharmacokinetics, safety and clinical use of linezolid in preterm infants. The Medline and EMBASE databases were searched using the term 'linezolid' combined with 'newborn', 'neonate', 'preterm' and 'premature' for papers published between January 1987 and June 2009. Studies reporting on a population including preterm infants and other age groups as well as case reports on preterm infants only were acceptable for analysis. Five studies and eight case reports were identified evaluating linezolid in preterm infants. A dosage regimen of 10mg/kg body weight given either orally or intravenously every 8h in infants aged >or=1 week and the same dose given every 12h in infants <1 week was shown to be safe and effective with a mean treatment duration of 10-28 days. In summary, linezolid was shown to be a safe and effective alternative to vancomycin in the treatment of infections with multiresistant Gram-positive pathogens in preterm infants.

The effects of mating and instrumental insemination on queen honey bee flight behaviour and gene expression.

Mating is fundamental to most organisms, although the physiological and transcriptional changes associated with this process have been largely characterized only in Drosophila melanogaster. In this study, we use honey bees as a model system because their queens undergo massive and permanent physiological and behavioural changes following mating. Previous studies have identified changes associated with the transition from a virgin queen to a fully mated, egg-laying queen. Here, we further uncouple the mating process to examine the effects of natural mating vs. instrumental insemination and saline vs. semen insemination. We observed effects on flight behaviour, vitellogenin expression and significant overlap in transcriptional profiles between our study and analogous studies in D. melanogaster, suggesting that some post-mating mechanisms are conserved across insect orders.

Cytotoxic action of Serratia marcescens hemolysin on human epithelial cells.

Incubation of human epithelial cells with nanomolar concentrations of chromatographically purified Serratia marcescens hemolysin (ShlA) caused irreversible vacuolation and subsequent lysis of the cells. Vacuolation differed from vacuole formation by Helicobacter pylori VacA. Sublytic doses of ShlA led to a reversible depletion of intracellular ATP. Restoration to the initial ATP level was presumably due to the repair of the toxin damage and was inhibited by cycloheximide. Pores formed in epithelial cells and fibroblasts without disruption of the plasma membrane, and the pores appeared to be considerably smaller than those observed in artificial lipid membranes and in erythrocytes and did not allow the influx of propidium iodide or trypan blue. All cytotoxic effects induced by isolated recombinant ShlA were also obtained with exponentially growing S. marcescens cells. The previously suggested role of the hemolysin in the pathogenicity of S. marcescens is supported by these data.

Extrachromosomal homologous DNA recombination in plant cells is fast and is not affected by CpG methylation.

Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the beta-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared.

Budd-Chiari syndrome in children: report of 22 cases.

Clinical, radiologic, and histologic features in 22 children with Budd-Chiari syndrome are reported. Three children had acute refractory ascites; all the others had hepatomegaly, which was detected either fortuitously or because of abdominal pain or distention. Results of liver function tests were normal or only moderately abnormal. In most cases a combination of ultrasonography and needle liver biopsy pointed to the diagnosis of Budd-Chiari syndrome, which was confirmed by angiography. Eighteen children underwent surgery involving various techniques, depending on the degree of patency of the inferior vena cava. Five children died postoperatively. Histologic studies of the liver, carried out in 12 of the surviving children, showed disappearance or regression of centrilobular hemorrhagic infiltration. Half of the surviving surgical patients are now free of complications after a follow-up of 7 months to 7 years; the others have either secondary thrombosis of the inferior vena cava or stenosis of the shunt or have experienced late pulmonary complications. Our results suggest that (1) Budd-Chiari syndrome must be considered a possible diagnosis in children with firm hepatomegaly and normal or near normal liver function, (2) surgery provides good results in many instances, and (3) the possibility of late complications requires careful follow-up.