Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium ...tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2-deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon gamma- and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and beta-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of sigmaE-dependent, sodium dodecyl sulfate-regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.
Comprehensive and systematic analysis of airway gene expression represents a strategy for addressing the multiple, complex, and largely untested hypotheses that exist for disease mechanisms, ...including asthma. Here, we report a novel real-time PCR-based method specifically designed for quantification of multiple low-abundance transcripts using as little as 2.5 fg of total RNA per gene. This method of gene expression profiling has the same specificity and sensitivity as RT-PCR and a throughput level comparable to low-density DNA microarray hybridization. In this two-step method, multiplex RT-PCR is successfully combined with individual gene quantification via real-time PCR on generated cDNA product. Using this method, we measured the expression of 75 genes in bronchial biopsies from asthmatic versus healthy subjects and found expected increases in expression levels of Th2 cytokines and their receptors in asthma. Surprisingly, we also found increased gene expression of NKCC1--a Na+-K+-Cl- cotransporter. Using immunohistochemical method, we confirmed increased protein expression for NKCC1 in the asthmatic subject with restricted localization to goblet cells. These data validate the new transcriptional profiling method and implicate NKCC1 in the pathophysiology of mucus hypersecretion in asthma. Potential applications for this method include transcriptional profiling in limited numbers of laser captured cells and validation of DNA microarray data in clinical specimens.
Proportional electroluminescence (EL) in noble gases is used in two-phase detectors for dark matter searches to record (in the gas phase) the ionization signal induced by particle scattering in the ...liquid phase. The “standard” EL mechanism is considered to be due to noble gas excimer emission in the vacuum ultraviolet (VUV). In addition, there are two alternative mechanisms, producing light in the visible and near infrared (NIR) ranges. The first is due to bremsstrahlung of electrons scattered on neutral atoms (“neutral bremsstrahlung”, NBrS). The second, responsible for electron avalanche scintillation in the NIR at higher electric fields, is due to transitions between excited atomic states. In this work, we have for the first time demonstrated two alternative techniques of the optical readout of two-phase argon detectors, in the visible and NIR range, using a silicon photomultiplier matrix and electroluminescence due to either neutral bremsstrahlung or avalanche scintillation. The amplitude yield and position resolution were measured for these readout techniques, which allowed to assess the detection threshold for electron and nuclear recoils in two-phase argon detectors for dark matter searches. To the best of our knowledge, this is the first practical application of the NBrS effect in detection science.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We have developed modifications to direct cDNA selection that allow the rapid and reproducible isolation of low abundance cDNAs encoded by large genomic clones. Biotinylated, cloned genomic DNAs are ...hybridized in solution with amplifiable cDNAs. The genomic clones and attached cDNAs are captured on streptavidin coated magnetic beads, the cDNAs are eluted and amplified. We have applied this protocol to a 425kb YAC that contains the human IL4 and IL5 genes. After two cycles of enrichment twenty-four cDNAs were evaluated, all of which were homologous to the YAC. DNA sequencing revealed that nine cDNAs were 100% homologous to the interferon regulatory factor 1 (IRF1) gene. Six clones were 70% homologous to the murine P600 gene, which is coexpressed with IL4 and IL5 in mouse Th2 cells. The nine remaining clones were unique within the sequence databases and were non redundant. All of the selected cDNAs were initially present at very low abundance and were enriched by as much as 100,000-fold in two cycles of enrichment. This modified selection technique should be readily applicable to the isolation of many candidate disease loci as well as the derivation of detailed transcription maps across large genomic regions.
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The article presents the results from putting a new automated flow traversing measurement system (AFTMS) in an experimental turbine intended for testing steam paths of the low-pressure cylinders ...(LPC) of large-capacity steam turbines into use. Modern computerized facilities have been used in developing the system. Such an approach has been implemented for the first time in the full-scale test bench available at PJSC Power Machines in carrying out experimental investigations of an LPC double stage compartment (on a 1 : 1 scale) equipped with a new, ultimately long last stage blade (LSB) developed for a steam turbine plant for ultrasupercritical steam conditions. The investigations were aimed at confirming the effectiveness of the developed system in measuring the flow parameters in the last LPC stage. The necessity of making a shift from nonautomated and semiautomatic systems to the use of automated measurement systems is substantiated. The article describes a system that makes it possible, with automated setting of the gas dynamic probe head position on the traversing line, to determine the steam flow direction; to measure the stagnation pressure, static pressure, and stagnation temperature; and to record the measures indicators with exactly assigned time tags. The results from measurements of flow parameters upstream of the last LPC stage in a partial load mode are given. A comparison between the experimental and calculated flow parameter distribution data over the stage radius in the control section is shown.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The Aria cryogenic distillation plant, located in Sardinia, Italy, is a key component of the DarkSide-20k experimental program for WIMP dark matter searches at the INFN Laboratori Nazionali del Gran ...Sasso, Italy. Aria is designed to purify the argon, extracted from underground wells in Colorado, USA, and used as the DarkSide-20k target material, to detector-grade quality. In this paper, we report the first measurement of argon isotopic separation by distillation with the 26 m tall Aria prototype. We discuss the measurement of the operating parameters of the column and the observation of the simultaneous separation of the three stable argon isotopes:
36
Ar
,
38
Ar
, and
40
Ar
. We also provide a detailed comparison of the experimental results with commercial process simulation software. This measurement of isotopic separation of argon is a significant achievement for the project, building on the success of the initial demonstration of isotopic separation of nitrogen using the same equipment in 2019.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A double-phase argon Time Projection Chamber (TPC), with an active mass of 185 g, has been designed and constructed for the Recoil Directionality (ReD) experiment. The aim of the ReD project is to ...investigate the directional sensitivity of argon-based TPCs via columnar recombination to nuclear recoils in the energy range of interest (20–
200
keV
nr
) for direct dark matter searches. The key novel feature of the ReD TPC is a readout system based on cryogenic Silicon Photomultipliers (SiPMs), which are employed and operated continuously for the first time in an argon TPC. Over the course of 6 months, the ReD TPC was commissioned and characterised under various operating conditions using
γ
-ray and neutron sources, demonstrating remarkable stability of the optical sensors and reproducibility of the results. The scintillation gain and ionisation amplification of the TPC were measured to be
g
1
=
(
0.194
±
0.013
)
photoelectrons/photon and
g
2
=
(
20.0
±
0.9
)
photoelectrons/electron, respectively. The ratio of the ionisation to scintillation signals (S2/S1), instrumental for the positive identification of a candidate directional signal induced by WIMPs, has been investigated for both nuclear and electron recoils. At a drift field of 183 V/cm, an S2/S1 dispersion of 12% was measured for nuclear recoils of approximately 60–
90
keV
nr
, as compared to 18% for electron recoils depositing 60 keV of energy. The detector performance reported here meets the requirements needed to achieve the principal scientific goals of the ReD experiment in the search for a directional effect due to columnar recombination. A phenomenological parameterisation of the recombination probability in LAr is presented and employed for modeling the dependence of scintillation quenching and charge yield on the drift field for electron recoils between 50–500 keV and fields up to 1000 V/cm.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
The specific activity of the
$$\beta $$
β
decay of
$$^{39}$$
39
Ar in atmospheric argon is measured using the DEAP-3600 detector. DEAP-3600, located 2 km underground at SNOLAB, uses a total ...of (3269 ± 24) kg of liquid argon distilled from the atmosphere to search for dark matter. This detector is well-suited to measure the decay of
$$^{39}$$
39
Ar owing to its very low background levels. This is achieved in two ways: it uses low background construction materials; and it uses pulse-shape discrimination to differentiate between nuclear recoils and electron recoils. With 167 live-days of data, the measured specific activity at the time of atmospheric extraction is (0.964 ± 0.001
$$_\textrm{stat}$$
stat
± 0.024
$$_\textrm{sys}$$
sys
) Bq/kg
$$_\textrm{atmAr}$$
atmAr
, which is consistent with results from other experiments. A cross-check analysis using different event selection criteria and a different statistical method confirms the result.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Integrins are heterodimeric cell-surface proteins that regulate cell growth, migration and survival. We have shown previously that the epithelial-restricted integrin alpha v beta 6 has another ...critical function; that is, it binds and activates latent transforming growth factor- beta (TGF- beta ). Through a global analysis of pulmonary gene expression in the lungs of mice lacking this integrin (Itgb6 null mice) we have identified a marked induction of macrophage metalloelastase (Mmp12): a metalloproteinase that preferentially degrades elastin and has been implicated in the chronic lung disease emphysema. Here we report that Itgb6-null mice develop age-related emphysema that is completely abrogated either by transgenic expression of versions of the beta 6 integrin subunit that support TGF- beta activation, or by the loss of Mmp12. Furthermore, we show that the effects of Itgb6 deletion are overcome by simultaneous transgenic expression of active TGF- beta 1. We have uncovered a pathway in which the loss of integrin-mediated activation of latent TGF- beta causes age-dependent pulmonary emphysema through alterations of macrophage Mmp12 expression. Furthermore, we show that a functional alteration in the TGF- beta activation pathway affects susceptibility to this disease.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
1 Cardiovascular Research Institute and 2 Department of Medicine/Physiology, University of California, San Francisco, California; and 3 Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, ...Genoa, Italy
Submitted 20 April 2005
; accepted in final form 29 August 2005
Previous studies in intact lung suggest that CFTR may play a role in cAMP-regulated fluid transport from the distal air spaces of the lung. However, the potential contribution of different epithelial cells (alveolar epithelial type I, type II, or bronchial epithelial cells) to CFTR-regulated fluid transport is unknown. In this study we determined whether the CFTR gene is expressed in human lung alveolar epithelial type II (AT II) cells and whether the CFTR chloride channel contributes to cAMP-regulated fluid transport in cultured human AT II cells. Human AT II cells were isolated and cultured on collagen I-coated Transwell membranes for 120144 h with an air-liquid interface. The cultured cells retained typical AT II-like features based on morphologic studies. Net basal fluid transport was 0.9 ± 0.1 µl·cm 2 ·h 1 and increased to 1.35 ± 0.11 µl·cm 2 ·h 1 (mean ± SE, n = 18, P < 0.05) by stimulation with cAMP agonists. The CFTR inhibitor, CFTR inh -172, inhibited cAMP stimulated but not basal fluid transport. In short-circuit current ( I sc ) studies with an apical-to-basolateral transepithelial Cl gradient, apical application of CFTR inh -172 reversed the forskolin-induced decrease in I sc . Real time RT-PCR demonstrated CFTR transcript expression in human AT II cells at a level similar to that in airway epithelial cells. We conclude that CFTR is expressed in cultured human AT II cells and may contribute to cAMP-regulated apical-basolateral fluid transport.
lung epithelial cells; cystic fibrosis transmembrane conductance regulator; ion channel; cyclic adenosine monophosphate; pulmonary edema; alveolar fluid clearance
Address for reprint requests and other correspondence: X. Fang, Cardiovascular Research Inst., Univ. of California, San Francisco, CA 94143-0130 (e-mail: xiaohui.fang{at}ucsf.edu )
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