Establishment of a positive-readout reporter system for siRNAs

The use of small interfering RNA molecules for therapeutic applications requires development of improved delivery systems, a process that would be facilitated by a non-invasive positive-readout mouse model for studying siRNA pharmacodynamics. Positive readout would yield better signal/noise ratios than existing negative-readout systems. We have engineered a positive-readout luciferase reporter system, activated by successful delivery of siRNA targeting the lac repressor. Co-transfection of a plasmid expressing lac repressor and a plasmid expressing firefly luciferase under the control of an RSV promoter, containing two lac operator sites, resulted in 5.7-fold lower luciferase activity than luciferase-encoding plasmid alone. Inhibition was reversed following addition of synthetic inducer, IPTG, which elevated luciferase expression to normal levels and confirmed functionality of the lac operon. Delivery of 1nM siRNA targeting lac repressor to repressor/reporter co-transfected cells was sufficient to fully restore luciferase expression to levels observed in the absence of repressor. Maximum expression was observed after 48hr, with a rapid decrease thereafter due to the short half life of luciferase. The luciferase positive-readout reporter system is therefore a dynamic indicator of successful RNAi delivery in vitro and could be adapted to generate a transgenic mouse capable of reporting RNAi activity non-invasively in vivo

Cancer stem cells: Mediators of tumorigenesis and metastasis in head and neck squamous cell carcinoma

BackgroundCancer stem cells (CSCs) represent a subpopulation of cells responsible for tumor growth. Their role in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and metastasis remains uncertain.MethodsWound healing and an orthotopic animal model were used to study cells expressing the CSC phenotype (CD44high and aldehyde dehydrogenase [ALDH]+) and assess mobility, tumorigenesis, and metastasis. A prospective collection of 40 patient‐derived primary HNSCC specimens were analyzed for CSC‐proportion compared to clinical variables.ResultsCSCs exhibited significantly faster wound closure and greater tumorigenesis and regional metastasis in vivo than non‐CSCs. In primary patient tumors, size and advanced stage were correlated with elevated proportion of CSCs, however, not with survival.ConclusionHNSCC stem cells mediate tumorigenesis and regional metastasis in vivo. In primary patient tumors, CSC‐proportion was associated with tumor size and stage, but not with metastatic spread or survival. CSC burden alone may only represent a minor variable in understanding CSCs and metastasis. © 2014 Wiley Periodicals, Inc. Head Neck 37: 317–326, 2015Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110728/1/hed23600.pd

The Fate of Sub-micron Circumplanetary Dust Grains II: Multipolar Fields

We study the radial and vertical stability of dust grains launched with all charge-to-mass ratios at arbitrary distances from rotating planets with complex magnetic fields. We show that the aligned dipole magnetic field model analyzed by Jontof-Hutter and Hamilton (2012) is an excellent approximation in most cases, but that fundamentally new physics arises with the inclusion of non-axisymmetric magnetic field terms. In particular, large numbers of distant negatively-charged dust grains, stable in a magnetic dipole, can be driven to escape by a more complex field. We trace the origin of the instability to overlapping Lorentz resonances which are extremely powerful when the gravitational and electromagnetic forces on a dust grain are comparable. These resonances enable a dust grain to tap the spin energy of the planet to power its escape. We also explore the relatively minor influence of different launch speeds and the far more important effects of variable grain charge. Only the latter are capable of significantly affecting the micron-sized grains that dominate visible and infrared images of faint dust rings. Finally, we present full stability maps for Earth, Jupiter, Saturn, Uranus, and Neptune with magnetic fields modeled out to octupole order. Not surprisingly, dust in the tortured magnetic fields of Uranus and Neptune show the greatest instability.Comment: 60 pages in manuscript format, 17 figures, 1 tabl

Near-Infrared Survey and Photometric Redshifts in the Extended GOODS-North field

We present deep $J$ and $H$-band images in the extended Great Observatories Origins Deep Survey-North (GOODS-N) field covering an area of 0.22 $\rm{deg}^{2}$. The observations were taken using WIRCam on the 3.6-m Canada France Hawaii Telescope (CFHT). Together with the reprocessed $K_{\rm s}$-band image, the $5\sigma$ limiting AB magnitudes (in 2" diameter apertures) are 24.7, 24.2, and 24.4 AB mag in the $J$, $H$, and $K_{\rm s}$ bands, respectively. We also release a multi-band photometry and photometric redshift catalog containing 93598 sources. For non-X-ray sources, we obtained a photometric redshift accuracy $\sigma_{\mathrm{NMAD}}=0.036$ with an outlier fraction $\eta = 7.3\%$. For X-ray sources, which are mainly active galactic nuclei (AGNs), we cross-matched our catalog with the updated 2M-CDFN X-ray catalog from Xue et al. (2016) and found that 658 out of 683 X-ray sources have counterparts. $GALEX$ UV data are included in the photometric redshift computation for the X-ray sources to give $\sigma_{\mathrm{NMAD}} = 0.040$ with $\eta=10.5\%$. Our approach yields more accurate photometric redshift estimates compared to previous works in this field. In particular, by adopting AGN-galaxy hybrid templates, our approach delivers photometric redshifts for the X-ray counterparts with fewer outliers compared to the 3D-HST catalog, which fit these sources with galaxy-only templates

Cryogenic 160-GHz MMIC Heterodyne Receiver Module

A cryogenic 160-GHz MMIC heterodyne receiver module has demonstrated a system noise temperature of 100 K or less at 166 GHz. This module builds upon work previously described in Development of a 150-GHz MMIC Module Prototype for Large-Scale CMB Radiation (NPO-47664), NASA Tech Briefs, Vol. 35, No. 8 (August 2011), p. 27. In the original module, the local oscillator signal was saturating the MMIC low-noise amplifiers (LNAs) with power. In order to suppress the local oscillator signal from reaching the MMIC LNAs, the W-band (75 110 GHz) signal had to be filtered out before reaching 140 170 GHz. A bandpass filter was developed to cover 120 170 GHz, using microstrip parallel-coupled lines to achieve the desired filter bandwidth, and ensure that the unwanted W-band local oscillator signal would be sufficiently suppressed. With the new bandpass filter, the entire receiver can work over the 140 180-GHz band, with a minimum system noise temperature of 460 K at 166 GHz. The module was tested cryogenically at 20 K ambient temperature, and it was found that the receiver had a noise temperature of 100 K over an 8-GHz bandwidth. The receiver module now includes a microstrip bandpass filter, which was designed to have a 3-dB bandwidth of approximately 120-170 GHz. The filter was fabricated on a 3-mil-thick alumina substrate. The filter design was based on a W-band filter design made at JPL and used in the QUIET (Q/U Imaging ExperimenT) radiometer modules. The W-band filter was scaled for a new center frequency of 150 GHz, and the microstrip segments were changed accordingly. Also, to decrease the bandwidth of the resulting scaled design, the center gaps between the microstrip lines were increased (by four micrometers in length) compared to the gaps near the edges. The use of the 150-GHz bandpass filter has enabled the receiver module to function well at room temperature. The system noise temperature was measured to be less than 600 K (at room temperature) from 154 to 168 GHz. Additionally, the use of a W-band isolator between the receiver module and the local oscillator source also improved the noise temperature substantially. This may be because the mixer was presented with a better impedance match with the use of the isolator. Cryogenic testing indicates a system noise temperature of 100 K or less at 166 GHz. Prior tests of the MMIC amplifiers alone have resulted in a system noise temperature of 65.70 K in the same frequency range (.160 GHz) when cooled to an ambient temperature of 20 K. While other detector systems may be slightly more sensitive (such as SIS mixers), they require more cooling (to 4 K ambient) and are not as easily scalable to build a large array, due to the need for large magnets and other equipment. When cooled to 20 K, this receiver module achieves approximately 100 K system noise temperature, which is slightly higher than single-amplifier module results obtained at JPL (65.70 K when an amplifier is corrected for back-end noise contributions). If this performance can be realized in practice, and a scalable array can be produced, the impact on cosmic microwave background experiments, astronomical and Earth spectroscopy, interferometry, and radio astronomy in general will be dramatic

Folliculin regulates cell–cell adhesion, AMPK, and mTORC1 in a cell‐type‐specific manner in lung‐derived cells

Abstract Germline loss‐of‐function BHD mutations cause cystic lung disease and hereditary pneumothorax, yet little is known about the impact of BHD mutations in the lung. Folliculin (FLCN), the product of the Birt–Hogg–Dube (BHD) gene, has been linked to altered cell–cell adhesion and to the AMPK and mTORC1 signaling pathways. We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho‐S6, a marker of mTORC1 activation, highlighting the cell type–dependent functions of FLCN. Cell–cell adhesion forces were significantly increased in FLCN‐deficient HBE cells, consistent with prior findings in FLCN‐deficient human kidney‐derived cells. To determine how these altered cell–cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd+/− mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg. Our results indicate that FLCN regulates the AMPK and mTORC1 pathways and cell–cell adhesion in a cell type–dependent manner. FLCN deficiency may impact the physiologic response to inflation‐induced mechanical stress, but further investigation is required. We hypothesize that FLCN‐dependent effects on signaling and cellular adhesion contribute to the pathogenesis of cystic lung disease in BHD patients

Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments

The American Association for the Surgery of Trauma renal injury grading scale: Implications of the 2018 revisions for injury reclassification and predicting bleeding interventions.

BackgroundIn 2018, the American Association for the Surgery of Trauma (AAST) published revisions to the renal injury grading system to reflect the increased reliance on computed tomography scans and non-operative management of high-grade renal trauma (HGRT). We aimed to evaluate how these revisions will change the grading of HGRT and if it outperforms the original 1989 grading in predicting bleeding control interventions.MethodsData on HGRT were collected from 14 Level-1 trauma centers from 2014 to 2017. Patients with initial computed tomography scans were included. Two radiologists reviewed the scans to regrade the injuries according to the 1989 and 2018 AAST grading systems. Descriptive statistics were used to assess grade reclassifications. Mixed-effect multivariable logistic regression was used to measure the predictive ability of each grading system. The areas under the curves were compared.ResultsOf the 322 injuries included, 27.0% were upgraded, 3.4% were downgraded, and 69.5% remained unchanged. Of the injuries graded as III or lower using the 1989 AAST, 33.5% were upgraded to grade IV using the 2018 AAST. Of the grade V injuries, 58.8% were downgraded using the 2018 AAST. There was no statistically significant difference in the overall areas under the curves between the 2018 and 1989 AAST grading system for predicting bleeding interventions (0.72 vs. 0.68, p = 0.34).ConclusionAbout one third of the injuries previously classified as grade III will be upgraded to grade IV using the 2018 AAST, which adds to the heterogeneity of grade IV injuries. Although the 2018 AAST grading provides more anatomic details on injury patterns and includes important radiologic findings, it did not outperform the 1989 AAST grading in predicting bleeding interventions.Level of evidencePrognostic and Epidemiological Study, level III

XMM-Newton spectroscopy of an X-ray selected sample of RL AGNs

This paper presents the X-ray spectroscopy of an X-ray selected sample of 25 radio-loud (RL) AGNs extracted from the XBSS sample. The main goal is to study the origin of the X-ray spectral differences usually observed between radio-loud and radio-quiet (RQ) AGNs. To this end, a comparison sample of 53 RQ AGNs has been also extracted from the same XBSS sample and studied together with the sample of RL AGNs. We have focused the analysis on the distribution of the X-ray spectral indices of the power-law component that models the large majority of the spectra in both samples. We find that the mean X-ray energy spectral index is very similar in the 2 samples and close to alpha_X~1. However, the intrinsic distribution of the spectral indices is significantly broader in the sample of RL AGNs. In order to investigate the origin of this difference, we have divided the RL AGNs into blazars and ``non-blazars'', on the basis of the available optical and radio information. We find strong evidence that the broad distribution observed in the RL AGN sample is mainly due to the presence of the blazars. Furthermore, within the blazar class we have found a link between the X-ray spectral index and the value of the radio-to-X-ray spectral index suggesting that the observed X-ray emission is directly connected to the emission of the relativistic jet. This trend is not observed among the ``non-blazars'' RL AGNs. This favours the hypothesis that, in these latter sources, the X-ray emission is not significantly influenced by the jet emission and it has probably an origin similar to the RQ AGNs. Overall, the results presented here indicate that the observed distribution of the X-ray spectral indices in a given sample of RL AGNs is strongly dependent on the amount of relativistic beaming present in the selected sources.Comment: 18 pages, 10 figures, accepted for publication in A&

Intersecting Brane Worlds -- A Path to the Standard Model ?

In this review we describe the general geometrical framework of brane world constructions in orientifolds of type IIA string theory with D6-branes wrapping 3-cycles in a Calabi-Yau 3-fold. These branes generically intersect in points on the internal space, and the patterns of intersections govern the chiral fermion spectra. We discuss how the open string spectra in intersecting brane models are constructed, how the Standard Model can be embedded, and also how supersymmetry can be realized in this class of string vacua. After the general considerations we specialize the discussion to the case of orbifold backgrounds with intersecting D6-branes and to the quintic Calabi-Yau manifold. Then, we discuss parts of the effective action of intersecting brane world models. Specifically we compute from the Born-Infeld action of the wrapped D-branes the tree-level, D-term scalar potential, which is important for the stability of the considered backgrounds as well as for questions related to supersymmetry breaking. Second, we review the recent computation concerning of gauge coupling unification and also of one-loop gauge threshold corrections in intersecting brane world models. Finally we also discuss some aspects of proton decay in intersecting brane world models.Comment: 31 pages, To appear in the proceedings of the RTN-workshop ``The quantum structure of spacetime and the geometric nature of fundamental interactions'', September 2003 in Copenhagen, revised version contains new refs and one corrected equatio