Free Molecule Studies by Perturbed γ-γ Angular Correlation: A New Path to Accurate Nuclear Quadrupole Moments.

Accurate nuclear quadrupole moment values are essential as benchmarks for nuclear structure models and for the interpretation of experimentally determined nuclear quadrupole interactions in terms of electronic and molecular structure. Here, we present a novel route to such data by combining perturbed γ-γ angular correlation measurements on free small linear molecules, realized for the first time within this work, with state-of-the-art ab initio electronic structure calculations of the electric field gradient at the probe site. This approach, also feasible for a series of other cases, is applied to Hg and Cd halides, resulting in Q(^{199}Hg,5/2^{-})=+0.674(17) b and Q(^{111}Cd,5/2^{+})=+0.664(7) b.

Annealing studies combined with low temperature emission Mössbauer spectroscopy of short-lived parent isotopes: Determination of local Debye-Waller factors.

An extension of the online implantation chamber used for emission Mössbauer Spectroscopy (eMS) at ISOLDE/CERN that allows for quick removal of samples for offline low temperature studies is briefly described. We demonstrate how online eMS data obtained during implantation at temperatures between 300 K and 650 K of short-lived parent isotopes combined with rapid cooling and offline eMS measurements during the decay of the parent isotope can give detailed information on the binding properties of the Mössbauer probe in the lattice. This approach has been applied to study the properties of Sn impurities in ZnO following implantation of 119In (T½ = 2.4 min). Sn in the 4+ and 2+ charge states is observed. Above T > 600 K, Sn2+ is observed and is ascribed to Sn on regular Zn sites, while Sn2+ detected at T < 600 K is due to Sn in local amorphous regions. A new annealing stage is reported at T ≈ 550 K, characterized by changes in the Sn4+ emission profile, and is attributed to the annihilation of close Frenkel pairs.

Local increase of the Curie temperature in Mn/Fe implanted Y3Fe5O12 (YIG).

The change in the Curie temperature of single crystalline garnet Y3Fe5O12 (YIG) sample due to lattice damage induced by ion implantation has been investigated in 57Fe emission Mössbauer Spectroscopy (eMS) following implantation of 57Mn (T½ = 1.5 min). The Mössbauer spectra analysis reveal high spin Fe3+ ions substituted on both the octahedral and the tetrahedral sites. Measurements in the temperature range 298 K-798 K show that average values of the magnetic hyperfine field are decreased by the implantation-induced damage on the local lattice structure of the YIG. The Curie temperature, however, is determined to be 651 ± 5 K, considerably higher than the value of bulk YIG (559 K). This is most likely due to lattice damage-induced changes on the spin configurations of YIG through a FeA-O-FeD distortion scheme.

Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development.

Early human development is a dynamic, heterogeneous, complex and multidimensional process. During the first week, the single-cell zygote undergoes eight to nine rounds of cell division generating the multicellular blastocyst, which consists of hundreds of cells forming spatially organized embryonic and extra-embryonic tissues. At the level of transcription, degradation of maternal RNA commences at around the two-cell stage, coinciding with embryonic genome activation. Although numerous efforts have recently focused on delineating this process in humans, many questions still remain as thorough investigation has been limited by ethical issues, scarce availability of human embryos and the presence of minute amounts of DNA and RNA. In vitro cultures of embryonic stem cells provide some insight into early human development, but such studies have been confounded by analysis on a population level failing to appreciate cellular heterogeneity. Recent technical developments in single-cell RNA sequencing have provided a novel and powerful tool to explore the early human embryo in a systematic manner. In this review, we will discuss the advantages and disadvantages of the techniques utilized to specifically investigate human development and consider how the technology has yielded new insights into pre-implantation development, embryonic stem cells and the establishment of the germ line.

Towards a Hall effect magnetic tracking device for MRI.

This paper presents the first prototype of a magnetic tracking device for Magnetic Resonance Imaging. The unique relationship between the space coordinates of a MRI scanner bore and the magnetic field gradients used in MRI allows building a localization system based on an accurate measurement of these gradients. These gradients are measured thanks to a 3D Hall device with a footprint of only 50µm(2), integrated with its specific conditioning circuit in a low cost, low voltage 0.35µm CMOS process. The first experimental results show that a sub-millimeter localization is possible. It opens the way to the development of MRI compatible magnetic tracking systems integrable in a surgical tool.

The structural transition of the prion protein into its pathogenic conformation is induced by unmasking hydrophobic sites.

A series of structural intermediates in the putative pathway from the cellular prion protein PrP(C) to the pathogenic form PrP(Sc) was established by systematic variation of low concentrations (<0.1%) of the detergent sodium dodecyl sulfate (SDS) or by the interaction with the bacterial chaperonin GroEL. Most extended studies were carried out with recombinant PrP (90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30. Similar results were obtained with full-length recombinant PrP, hamster PrP 27-30 and PrP(C) isolated from transgenic, non-infected CHO cells. Varying the incubation conditions, i.e. the concentration of SDS, the GroEL and GroEL/ES, but always at neutral pH and room temperature, different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and to molecular mass, as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation: alpha-helical monomers, soluble alpha-helical dimers, soluble but beta-structured oligomers of a minimal size of 12-14 PrP molecules, and insoluble multimers were observed. A high activation barrier was found between the alpha-helical dimers and beta-structured oligomers. The numbers of SDS-molecules bound to PrP in different conformations were determined: Partially denatured, alpha-helical monomers bind 31 SDS molecules per PrP molecule, alpha-helical dimers 21, beta-structured oligomers 19-20, and beta-structured multimers show very strong binding of five SDS molecules per PrP molecule. Binding of only five molecules of SDS per molecule of PrP leads to fast formation of beta-structures followed by irreversible aggregation. It is discussed that strongest binding of SDS has an effect identical with or similar to the interaction with GroEL thereby inducing identical or very similar transitions. The interaction with GroEL/ES stabilizes the soluble, alpha-helical conformation. The structure and their stabilities and particularly the induction of transitions by interaction of hydrophobic sites of PrP are discussed in respect to their biological relevance.

Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression.

The suitability of the recently described red fluorescent protein dsRED from reef corals for use as a reporter in plant molecular biology was investigated. Based on the clone pDSRED (Clontech), plant expression vectors were constructed for constitutive dsRED expression in the cytosol, the endoplasmic reticulum and the vacuole. Fluorescence microscopy of tobacco BY2 suspension culture cells transiently expressing the plant vectors generated proved that cytosolic expression of the dsRED gives rise to readily detectable levels of red fluorescence, whereas expression in the ER was poor. Vacuolar dsRED expression did not result in any significant fluorescence. dsRED transgenic tobacco SR1 plants were generated to test the sensitivity of dsRED as a reporter in an autofluorescent background, and to identify the possible impact of the introduced fluorescent protein on morphogenesis, plant development and fertility. During the transformation and regeneration phase plants did not show any abnormalities, indicating that dsRED is not interfering with plant development and morphogenesis. Regenerated plants were analysed by PCR, Western blot and fluorescence microscopy for the presence and expression of the transferred genes. The filter sets chosen for fluorescence microscopy proved to be able to block the red chlorophyll fluorescence completely, allowing specific dsRED detection. Best expression levels were obtained with dsRED targeted to the cytosol or chloroplasts. ER-targeted expression of dsRED also gave rise to readily detectable fluorescence levels, whereas vacuolar expression yielded no fluorescence. dsRED transgenic plant lines expressing the protein in the cytosol, ER or chloroplast proved to be fertile. Seed set and germination were normal, except that the seeds and seedlings maintained the red fluorescence phenotype.

Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells.

Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.

PCBs and neurodevelopmental effects in Michigan children: an evaluation of exposure and dose characterization.

Despite the fact that PCB levels in the general environment have continued to decline over the past decade, concern for potential neurodevelopmental deficits from in utero exposure to these compounds remains unabated. In fact, some regulatory and scientific bodies have concluded that the evidence suggesting that prenatal exposure to PCBs may lead to neurodevelopmental deficits is one of the greatest public health concerns surrounding PCBs. The primary basis for the concern that low-level in utero exposure to PCBs causes neurodevelopmental deficits in children is a series of reports on a cohort of Michigan children presumably exposed to PCBs as a result of their mother's consumption of Great Lakes fish. These children, known collectively as the Jacobson cohort, have been followed from birth to 11 years of age. The investigators following these children concluded that they have demonstrated persistent neurodevelopmental effects in this cohort attributable solely to PCBs. However, a detailed analysis of the cohort's exposure characterization, particularly in the initial reports, reveals considerable uncertainty as to the actual exposure status of mothers characterized as "fish eaters" and their offspring. Failure to adequately characterize the PCB exposure of these mothers, or their children, precludes any causal association between in utero exposure to PCBs and neurodevelopmental deficits.

SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase.

Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.

Differential effects of adult and perinatal lead exposure on morphine-induced locomotor activity in rats.

The effects of adult and perinatal lead treatment on the development of locomotor sensitization produced with repeated morphine administration was investigated. In Experiment 1, adult male rats received a diet containing 250 ppm lead acetate or a control diet for 43 days. Animals then received 10 mg/kg morphine sulfate or water vehicle (ip) and locomotor activity was monitored for 14 consecutive days. While both control and lead-exposed animals demonstrated a locomotor sensitization to morphine, the magnitude of the increased locomotor response was reduced in lead-treated animals. Subsequent analysis of blood-lead in the adult lead-exposed animals indicated residue levels ranging between 20 and 30 microg/dl. In Experiment 2, adult female rats were treated daily with 0, 8, or 16 mg lead via gavage for 30 days before breeding with non-exposed males. Lead exposure in dams continued through gestation and until pups were weaned at postnatal day (PND) 21. At PND 60, male offspring received morphine or vehicle challenges identical to those described in Experiment 1. Animals perinatally exposed to dams receiving 16 mg lead daily demonstrated an enhanced behavioral response to morphine relative to control animals. Analysis of offspring blood indicated lead levels below detectable limits (<1 microg/dl) for all animals. The results suggest exposure to lead at environmentally relevant levels produces long-lasting changes in drug-induced behavior, and the developmental period in which lead exposure occurs is a significant contributor to the manifestation of these effects.

Intron-tagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells.

Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo. Using T-DNA-mediated transformation, the detection of epitope-tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope-tagged proteins in Agrobacterium-infected plant cells. We have developed a method for labelling plant coding sequences with intron-tagged epitope-coding domains that are not processed in Agrobacterium. Here we show that the expression of HA-epitope-tagged constructs encoding beta-glucuronidase and S-phase kinase-associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection. This epitope-tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium-transformed plant cells.

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium.

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Survival analysis in quality improvement: the Diabetic Kidney Disease Project extrapolation group estimates.

Survival analysis is used in a wide variety of research settings to maximize the information extracted from a group of timed observations. Measures employed in continuous quality improvement (CQI) efforts often involve such observations. Yet to date, survival analysis has not been widely used to guide CQI efforts. This article presents the features of survival analysis that are most applicable to CQI efforts and illustrates the application of these techniques to a quality improvement project focused on diabetic kidney disease. Results are compared with those from a "standard" analysis. The interpretation of results is discussed in the context of constraints typical of CQI efforts. The article concludes with a recommendation for broader application of this valuable analytic methodology.

Detection of point mutations by capillary electrophoresis with temporal temperature gradients.

By combining the advantages of capillary electrophoresis and temperature gradient gel electrophoresis, a method was developed to detect point mutations in polymerase chain reaction (PCR) fragments. Increasing and decreasing temporal temperature gradients were established by means of a computer-controlled Peltier module. Native and denaturing conditions were achieved by cooling to 25 degrees C and heating to 70 degrees C, respectively, a thermostating liquid surrounding the capillary. To separate nucleic acid fragments, a sieving media, containing 4% linear polyacrylamide, 1 x Tris borate EDTA buffer (TBE) and 6 M urea, was found appropriate. Renewal of the sieving matrix before each run significantly improved the reproducibility of fragment separation. The ability of this capillary electrophoresis system to detect point mutations is demonstrated with the human prion-protein gene.

Regulatory interaction of PRL1 WD protein with Arabidopsis SNF1-like protein kinases.

Mutation of the PRL1 gene, encoding a regulatory WD protein, results in glucose hypersensitivity and derepression of glucose-regulated genes in Arabidopsis. The yeast SNF1 protein kinase, a key regulator of glucose signaling, and Arabidopsis SNF1 homologs AKIN10 and AKIN11, which can complement the Deltasnf1 mutation, were found to interact with an N-terminal domain of the PRL1 protein in the two-hybrid system and in vitro. AKIN10 and AKIN11 suppress the yeast Deltasnf4 mutation and interact with the SNF4p-activating subunit of SNF1. PRL1 and SNF4 bind independently to adjacent C-terminal domains of AKIN10 and AKIN11, and these protein interactions are negatively regulated by glucose in yeast. AKIN10 and AKIN11, purified in fusion with glutathione S-transferase, undergo autophosphorylation and phosphorylate a peptide of sucrose phosphate synthase in vitro. The sucrose phosphate synthase-peptide kinase activity of AKIN complexes detected by immunoprecipitation is stimulated by sucrose in light-grown Arabidopsis plants. In comparison with wild type, the activation level of AKIN immunocomplexes is higher in the prl1 mutant, suggesting that PRL1 is a negative regulator of Arabidopsis SNF1 homologs. This conclusion is supported by the observation that PRL1 is an inhibitor of AKIN10 and AKIN11 in vitro.

An evaluation of modeled benzene exposure and dose estimates published in the Chinese-National Cancer Institute collaborative epidemiology studies.

Risk estimates and cause and effect determinations are directly dependent on exposure and dose-response relationships. Recently, relative risks and excess cancer mortality attributed to occupational benzene exposure have been published in collaborative studies conducted by Chinese investigators and scientists from the National Cancer Institute. The results of these studies suggest increased risk of acute nonlymphocytic leukemia at relatively low benzene concentrations and associations with cancers not previously associated with benzene exposure. These studies are potentially important due to their size and potential to more thoroughly investigate the link between benzene exposure and cancer. However, there are questions concerning the validity of exposure and dose estimates supporting relative risk characterizations in these studies. Apparent discrepancies between modeled exposure and dose estimates and sources of actual measured exposure information and clinical markers of benzene toxicity raise serious concerns questioning the reliability of relative risk and cancer associations stated in these studies.