Recombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red homologous recombination protein Beta in Escherichia coli has approached ...100% efficiency. While highly efficient in E. coli, recombineering using the Red Synaptase/Exonuclease pair (SynExo) in other organisms declines in efficiency roughly correlating with phylogenetic distance from E. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstituted in vitro and shown to catalyze a model recombination reaction. Here we describe stimulation of gene targeting to edit a novel fluorescent protein gene in the human genome using ICP8 and compared its efficiency to that of a "humanized" version of Beta protein from phage λ. ICP8 significantly enhanced gene targeting rates in HEK 293T cells while Beta was not only unable to catalyze recombineering but inhibited gene targeting using endogenous recombination functions, despite both synaptases being well-expressed and localized to the nucleus. This proof of concept encourages developing species-specific SynExo recombinases for genome engineering.
Background Anodal transcranial direct current stimulation (tDCS) of the prefrontal cortex has been proposed as therapeutic intervention in major depression. According to clinical needs, this study ...addresses the question whether tDCS is effective in treatment resistant major depressive episodes. Methods Twenty-two patients with a major depressive episode were randomly assigned to a cross-over protocol comparing tDCS and placebo stimulation add-on to a stable antidepressant medication. The parameters of active tDCS were: 1 or 2 mA for 20 minutes/day, anode over the left dorsolateral prefrontal cortex, cathode over the contralateral supraorbital region. Active and placebo tDCS was applied for 2 weeks using indistinguishable DC stimulators. Patients, raters, and operators were blinded to treatment conditions. Results There was no significant difference in depression scores after 2 weeks of real compared with 2 weeks of sham tDCS. Scores on the Hamilton Depression Rating Scale were reduced from baseline by 14.7% for active tDCS and 10% for placebo tDCS. In contrast, subjective mood ratings showed an increase in positive emotions after real tDCS compared with sham tDCS. Conclusions Anodal tDCS, applied for 2 weeks, was not superior to placebo treatment in patients with treatment resistant depression. However, secondary outcome measures are pointing to a positive effect of tDCS on emotions. Therefore, modified and improved tDCS protocols should be carried out in controlled pilot trials to develop tDCS towards an efficacious antidepressant intervention in therapy-resistant depression.
The mRNA of the ubiquitin-like modifier FAT10 has been reported to be overexpressed in 90% of hepatocellular carcinoma (HCC) and in over 80% of colon, ovary and uterus carcinomas. Elevated FAT10 ...expression in malignancies was attributed to transcriptional upregulation upon the loss of p53. Moreover, FAT10 induced chromosome instability in long-term in vitro culture, which led to the hypothesis that FAT10 might be involved in carcinogenesis. In this study we show that interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha synergistically upregulated FAT10 expression in liver and colon cancer cells 10- to 100-fold. Real-time RT-PCR revealed that FAT10 mRNA was significantly overexpressed in 37 of 51 (72%) of human HCC samples and in 8 of 15 (53%) of human colon carcinomas. The FAT10 cDNA sequences in HCC samples were not mutated and intact FAT10 protein was detectable. FAT10 expression in both cancer tissues correlated with expression of the IFN-gamma- and TNF-alpha-dependent proteasome subunit LMP2 strongly suggesting that proinflammatory cytokines caused the joint overexpression of FAT10 and LMP2. NIH3T3 transformation assays revealed that FAT10 had no transforming capability. Taken together, FAT10 qualifies as a marker for an interferon response in HCC and colon carcinoma but is not significantly overexpressed in cancers lacking a proinflammatory environment.
The micro-orifice uniform deposit impactor–droplet freezing technique (MOUDI-DFT) combines particle collection by inertial impaction (via the MOUDI) and a microscope-based immersion freezing ...apparatus (the DFT) to measure atmospheric concentrations of ice nucleating particles (INPs) as a function of size and temperature. In the first part of this study we improved upon this recently introduced technique. Using optical microscopy, we investigated the non-uniformity of MOUDI aerosol deposits at spatial resolutions of 1, 0.25 mm, and for some stages when necessary 0.10 mm. The results from these measurements show that at a spatial resolution of 1 mm and less, the concentration of particles along the MOUDI aerosol deposits can vary by an order of magnitude or more. Since the total area of a MOUDI aerosol deposit ranges from 425 to 605 mm2 and the area analyzed by the DFT is approximately 1.2 mm2, this non-uniformity needs to be taken into account when using the MOUDI-DFT to determine atmospheric concentrations of INPs. Measurements of the non-uniformity of the MOUDI aerosol deposits were used to select positions on the deposits that had relatively small variations in particle concentration and to build substrate holders for the different MOUDI stages. These substrate holders improve reproducibility by holding the substrate in the same location for each measurement and ensure that DFT analysis is only performed on substrate regions with relatively small variations in particle concentration. In addition, the deposit non-uniformity was used to determine correction factors that take the non-uniformity into account when determining atmospheric concentrations of INPs. In the second part of this study, the MOUDI-DFT utilizing the new substrate holders was compared to the continuous flow diffusion chamber (CFDC) technique of Colorado State University. The intercomparison was done using INP concentrations found by the two instruments during ambient measurements of continental aerosols. Results from two sampling periods were compared, and the INP concentrations determined by the two techniques agreed within experimental uncertainty. The agreement observed here is commensurate with the level of agreement found in other studies where CFDC results were compared to INP concentrations measured with other methods.
RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, ...control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.
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•RalGPS2 is expressed in 5637 cancer cells and is essential for their growth.•RalGPS2 is required for TNT formation.•The overexpression of RalGPS2 PH domain markedly increased the number and length of TNT.•Interaction between RalA and Sec5 is required for TNT formation in 5637 cells.•RalGPS2 interacts with LST1 and RalA leading to TNT formation.
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Stem cells combined with biodegradable injectable scaffolds releasing growth factors hold great promises in regenerative medicine, particularly in the treatment of neurological ...disorders. We here integrated human marrow-isolated adult multilineage-inducible (MIAMI) stem cells and pharmacologically active microcarriers (PAMs) into an injectable non-toxic silanized-hydroxypropyl methylcellulose (Si-HPMC) hydrogel. The goal is to obtain an injectable non-toxic cell and growth factor delivery device. It should direct the survival and/or neuronal differentiation of the grafted cells, to safely transplant them in the central nervous system, and enhance their tissue repair properties. A model protein was used to optimize the nanoprecipitation conditions of the neuroprotective brain-derived neurotrophic factor (BDNF). BDNF nanoprecipitate was encapsulated in fibronectin-coated (FN) PAMs and the in vitro release profile evaluated. It showed a prolonged, bi-phasic, release of bioactive BDNF, without burst effect. We demonstrated that PAMs and the Si-HPMC hydrogel increased the expression of neural/neuronal differentiation markers of MIAMI cells after 1week. Moreover, the 3D environment (PAMs or hydrogel) increased MIAMI cells secretion of growth factors (b-NGF, SCF, HGF, LIF, PlGF-1, SDF-1α, VEGF-A & D) and chemokines (MIP-1α & β, RANTES, IL-8). These results show that PAMs delivering BDNF combined with Si-HPMC hydrogel represent a useful novel local delivery tool in the context of neurological disorders. It not only provides neuroprotective BDNF but also bone marrow-derived stem cells that benefit from that environment by displaying neural commitment and an improved neuroprotective/reparative secretome. It provides preliminary evidence of a promising pro-angiogenic, neuroprotective and axonal growth-promoting device for the nervous system.
Combinatorial tissue engineering strategies for the central nervous system are scarce. We developed and characterized a novel injectable non-toxic stem cell and protein delivery system providing regenerative cues for central nervous system disorders. BDNF, a neurotrophic factor with a wide-range effect, was nanoprecipitated to maintain its structure and released in a sustained manner from novel polymeric microcarriers. The combinatorial 3D support, provided by fibronectin-microcarriers and the hydrogel, to the mesenchymal stem cells guided the cells towards a neuronal differentiation and enhanced their tissue repair properties by promoting growth factors and cytokine secretion. The long-term release of physiological doses of bioactive BDNF, combined to the enhanced secretion of tissue repair factors from the stem cells, constitute a promising therapeutic approach.
For almost two decades, the airborne Fast In-situ Stratospheric Hygrometer (FISH) has stood for accurate and precise measurements of total water mixing ratios (WMR, gas phase + evaporated ice) in the ...upper troposphere and lower stratosphere (UT/LS). Here, we present a comprehensive review of the measurement technique (Lyman- alpha photofragment fluorescence), calibration procedure, accuracy and reliability of FISH. Crucial for FISH measurement quality is the regular calibration to a water vapor reference, namely the commercial frost-point hygrometer DP30. In the frame of this work this frost-point hygrometer is compared to German and British traceable metrological water standards and its accuracy is found to be 2-4 %. Overall, in the range from 4 to 1000 ppmv, the total accuracy of FISH was found to be 6-8 %, as stated in previous publications. For lower mixing ratios down to 1 ppmv, the uncertainty reaches a lower limit of 0.3 ppmv. For specific, non-atmospheric conditions, as set in experiments at the AIDA chamber - namely mixing ratios below 10 and above 100 ppmv in combination with high- and low-pressure conditions - the need to apply a modified FISH calibration evaluation has been identified. The new evaluation improves the agreement of FISH with other hygrometers to plus or minus 10 % accuracy in the respective mixing ratio ranges. Furthermore, a quality check procedure for high total water measurements in cirrus clouds at high pressures (400-500 hPa) is introduced. The performance of FISH in the field is assessed by reviewing intercomparisons of FISH water vapor data with other in situ and remote sensing hygrometers over the last two decades. We find that the agreement of FISH with the other hygrometers has improved over that time span from overall up to plus or minus 30 % or more to about plus or minus 5-20 % at < 10 ppmv and to plus or minus 0-15 % at > 10 ppmv. As presented here, the robust and continuous calibration and operation procedures of the FISH instrument over the last two decades establish the position of FISH as one of the core instruments for in situ observations of water vapor in the UT/LS.
Eph receptors belong to a subfamily of receptor tyrosine kinases that are activated by membrane-spanning ligands called ephrins. Previously, we demonstrated that the ephrinB1-EphB2 interaction ...regulates odontogenic/osteogenic differentiation from dental pulp cells (DPCs) in vitro. The goal of this study was to identify the molecular mechanisms regulated by the EphB2/ephrinB1 system that govern tertiary dentin formation in vitro and in vivo. During tooth development, ephrinB1, and EphB2 were expressed in preodontoblast and odontoblasts at postnatal day 4. EphrinB1 was continuously expressed in odontoblasts and odontoblastic processes until the completion of tooth eruption. In addition, ephrinB1 was expressed in odontoblastic processes 2 wk following tooth injury without pulp exposure, whereas EphB2 was expressed in the center of pulp niches but not odontoblasts. In a model of tooth injury with pulp exposure, ephrinB1 was strongly expressed in odontoblasts 4 wk postinjury. In vitro studies with human and mouse DPCs treated with calcium hydroxide (CH) or mineral trioxide aggregate (MTA) showed an increased expression of insulin-like growth factor 1 (IGF-1). Experiments using several inhibitors of IGF-1 receptor signaling revealed that inhibiting the Ras/Raf-1/MAPK pathway inhibited EphB2 expression, and inhibiting the PI3K/Akt/mTOR pathway specifically inhibited ephrinB1 gene expression. Tooth injury in mice with odontoblast-specific IGF-1 receptor ablation exhibited a reduced tertiary dentin volume, mineral density, and ephrinB1 expression 4 wk following injury. We conclude that the IGF-1/ephrinB1 axis plays significant roles in the early stages of tooth injury. Further research is needed to fully understand the potential of targeting ephrinB1 as a regenerative pulp therapy.
In situ measurements of total water have been obtained during several airborne field experiments in the Arctic (POLSTAR 1997 and 1998; EUPLEX/ENVISAT 2003), at midlatitudes (ENVISAT 2002, Cirrus 2003 ...and 2004, TROCCINOX 2005), and in the tropics (APE‐THESEO 1999, TROCCINOX/ENVISAT 2005, SCOUT‐O3 2005) in 52 flights in cirrus using the Jülich Lyman‐α fluorescence hygrometer FISH. For a subset of 28 flights, the measurements are complemented by gas phase measurements of H2O. From the data set obtained in these experiments, the ice water content (IWC) in cirrus clouds is derived using two different approaches and functions of the minimum, mean, median, and maximum IWC are provided. The data are analyzed as a function of temperature in the range 183–250 K for Arctic, midlatitudinal, and tropical regions thus extending previous climatologies to much lower temperatures and lower detectable IWC. For each temperature, IWC covers a broad band, decreasing with temperature over the whole temperature range. In the tropics, several events of enhanced ice water content are observed which are related to recent impact of convection.
Convective redistribution of ozone and its precursors between the boundary layer (BL) and the free troposphere (FT) influences photochemistry, in particular in the middle and upper troposphere (UT). ...We present a case study of convective transport during the GABRIEL campaign over the tropical rain forest in Suriname in October 2005. During one measurement flight the inflow and outflow regions of a cumulonimbus cloud (Cb) have been characterized. We identified a distinct layer between 9 and 11 km altitude with enhanced mixing ratios of CO, O3, HOx, acetone and acetonitrile. The elevated O3 contradicts the expectation that convective transport brings low-ozone air from the boundary layer to the outflow region. Entrainment of ozone-rich air is estimated to account for 62% (range: 33–91%) of the observed O3. Ozone is enhanced by only 5–6% by photochemical production in the outflow due to enhanced NO from lightning, based on model calculations using observations including the first reported HOx measurements over the tropical rainforest. The "excess" ozone in the outflow is most probably due to direct production by corona discharge associated with lightning. We deduce a production rate of 5.12 × 1028 molecules O3 flash−1 (range: 9.89 × 1026–9.82 × 1028 molecules O3 flash−1), which is at the upper limit of the range reported previously.
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