27 research outputs found
Subtoxic Alterations in Hepatocyte-Derived Exosomes: An Early Step in Drug-Induced Liver Injury?
Drug-induced liver injury (DILI) is a significant clinical and economic problem in the United States, yet the mechanisms that underlie DILI remain poorly understood. Recent evidence suggests that signaling molecules released by stressed hepatocytes can trigger immune responses that may be common across DILI mechanisms. Extracellular vesicles released by hepatocytes, principally hepatocyte-derived exosomes (HDEs), may constitute one such signal. To examine HDE alterations as a function of drug-induced stress, this work utilized prototypical hepatotoxicant acetaminophen (APAP) in male Sprague-Dawley (SD) rats, SD rat hepatocytes, and primary human hepatocytes. HDE were isolated using ExoQuick precipitation reagent and analyzed by quantification of the liver-specific RNAs albumin and microRNA-122 (miR-122). In vivo, significant elevations in circulating exosomal albumin mRNA were observed at subtoxic APAP exposures. Significant increases in exosomal albumin mRNA were also observed in primary rat hepatocytes at subtoxic APAP concentrations. In primary human hepatocytes, APAP elicited increases in both exosomal albumin mRNA and exosomal miR-122 without overt cytotoxicity. However, the number of HDE produced in vitro in response to APAP did not increase with exosomal RNA quantity. We conclude that significant drug-induced alterations in the liver-specific RNA content of HDE occur at subtoxic APAP exposures in vivo and in vitro, and that these changes appear to reflect selective packaging rather than changes in exosome number. The current findings demonstrate that translationally relevant HDE alterations occur in the absence of overt hepatocellular toxicity, and support the hypothesis that HDE released by stressed hepatocytes may mediate early immune responses in DILI
Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity
Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxinand model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses
Structural and Functional Analysis of the Human Nuclear Xenobiotic Receptor PXR in Complex with RXRα
The human nuclear xenobiotic receptor PXR recognizes a range of potentially harmful drugs and endobiotic chemicals, but must complex with the nuclear receptor RXRα to control the expression of numerous drug metabolism genes. To date, the structural basis and functional consequences of this interaction have remained unclear. Here we present 2.8 Å resolution crystal structures of the heterodimeric complex formed between the ligand binding domains (LBDs) of human PXR and RXRα. These structures establish that PXR and RXRα form a heterotetramer unprecedented in the nuclear receptor family of ligand-regulated transcription factors. We further show that both PXR and RXRα bind to the transcriptional coregulator SRC-1 with higher affinity when they are part of the PXR-RXRα heterotetramer complex than they do when each LBD is examined alone. Furthermore, we purify the full-length forms of each receptor from recombinant bacterial expression systems, and characterize their interactions with a range of direct and everted repeat DNA elements. Taken together, these data advance our understanding of PXR, the master regulator of drug metabolism gene expression in humans, in its functional partnership with RXRα
A cluster randomized controlled trial of the effectiveness and cost-effectiveness of Intermediate Care Clinics for Diabetes (ICCD) : study protocol for a randomized controlled trial
Background
World-wide healthcare systems are faced with an epidemic of type 2 diabetes. In the United Kingdom, clinical care is primarily provided by general practitioners (GPs) rather than hospital specialists. Intermediate care clinics for diabetes (ICCD) potentially provide a model for supporting GPs in their care of people with poorly controlled type 2 diabetes and in their management of cardiovascular risk factors. This study aims to (1) compare patients with type 2 diabetes registered with practices that have access to an ICCD service with those that have access only to usual hospital care; (2) assess the cost-effectiveness of the intervention; and (3) explore the views and experiences of patients, health professionals and other stakeholders.
Methods/Design
This two-arm cluster randomized controlled trial (with integral economic evaluation and qualitative study) is set in general practices in three UK Primary Care Trusts. Practices are randomized to one of two groups with patients referred to either an ICCD (intervention) or to hospital care (control).
Intervention group: GP practices in the intervention arm have the opportunity to refer patients to an ICCD - a multidisciplinary team led by a specialist nurse and a diabetologist. Patients are reviewed and managed in the ICCD for a short period with a goal of improving diabetes and cardiovascular risk factor control and are then referred back to practice.
or
Control group: Standard GP care, with referral to secondary care as required, but no access to ICCD.
Participants are adults aged 18 years or older who have type 2 diabetes that is difficult for their GPs to control. The primary outcome is the proportion of participants reaching three risk factor targets: HbA1c (≤7.0%); blood pressure (<140/80); and cholesterol (<4 mmol/l), at the end of the 18-month intervention period. The main secondary outcomes are the proportion of participants reaching individual risk factor targets and the overall 10-year risks for coronary heart disease(CHD) and stroke assessed by the United Kingdom Prospective Diabetes Study (UKPDS) risk engine. Other secondary outcomes include body mass index and waist circumference, use of medication, reported smoking, emotional adjustment, patient satisfaction and views on continuity, costs and health related quality of life. We aimed to randomize 50 practices and recruit 2,555 patients
Two Earth-sized planets orbiting Kepler-20
Since the discovery of the first extrasolar giant planets around Sun-like
stars, evolving observational capabilities have brought us closer to the
detection of true Earth analogues. The size of an exoplanet can be determined
when it periodically passes in front of (transits) its parent star, causing a
decrease in starlight proportional to its radius. The smallest exoplanet
hitherto discovered has a radius 1.42 times that of the Earth's radius (R
Earth), and hence has 2.9 times its volume. Here we report the discovery of two
planets, one Earth-sized (1.03R Earth) and the other smaller than the Earth
(0.87R Earth), orbiting the star Kepler-20, which is already known to host
three other, larger, transiting planets. The gravitational pull of the new
planets on the parent star is too small to measure with current
instrumentation. We apply a statistical method to show that the likelihood of
the planetary interpretation of the transit signals is more than three orders
of magnitude larger than that of the alternative hypothesis that the signals
result from an eclipsing binary star. Theoretical considerations imply that
these planets are rocky, with a composition of iron and silicate. The outer
planet could have developed a thick water vapour atmosphere.Comment: Letter to Nature; Received 8 November; accepted 13 December 2011;
Published online 20 December 201
Masses, radii, and orbits of small Kepler planets : The transition from gaseous to rocky planets
We report on the masses, sizes, and orbits of the planets orbiting 22 Kepler stars. There are 49 planet candidates around these stars, including 42 detected through transits and 7 revealed by precise Doppler measurements of the host stars. Based on an analysis of the Kepler brightness measurements, along with high-resolution imaging and spectroscopy, Doppler spectroscopy, and (for 11 stars) asteroseismology, we establish low false-positive probabilities (FPPs) for all of the transiting planets (41 of 42 have an FPP under 1%), and we constrain their sizes and masses. Most of the transiting planets are smaller than three times the size of Earth. For 16 planets, the Doppler signal was securely detected, providing a direct measurement of the planet's mass. For the other 26 planets we provide either marginal mass measurements or upper limits to their masses and densities; in many cases we can rule out a rocky composition. We identify six planets with densities above 5 g cm-3, suggesting a mostly rocky interior for them. Indeed, the only planets that are compatible with a purely rocky composition are smaller than 2 R ⊕. Larger planets evidently contain a larger fraction of low-density material (H, He, and H2O).Peer reviewedFinal Accepted Versio
Planetary Candidates Observed by Kepler, III: Analysis of the First 16 Months of Data
New transiting planet candidates are identified in sixteen months (May 2009 -
September 2010) of data from the Kepler spacecraft. Nearly five thousand
periodic transit-like signals are vetted against astrophysical and instrumental
false positives yielding 1,091 viable new planet candidates, bringing the total
count up to over 2,300. Improved vetting metrics are employed, contributing to
higher catalog reliability. Most notable is the noise-weighted robust averaging
of multi-quarter photo-center offsets derived from difference image analysis
which identifies likely background eclipsing binaries. Twenty-two months of
photometry are used for the purpose of characterizing each of the new
candidates. Ephemerides (transit epoch, T_0, and orbital period, P) are
tabulated as well as the products of light curve modeling: reduced radius
(Rp/R*), reduced semi-major axis (d/R*), and impact parameter (b). The largest
fractional increases are seen for the smallest planet candidates (197% for
candidates smaller than 2Re compared to 52% for candidates larger than 2Re) and
those at longer orbital periods (123% for candidates outside of 50-day orbits
versus 85% for candidates inside of 50-day orbits). The gains are larger than
expected from increasing the observing window from thirteen months (Quarter 1--
Quarter 5) to sixteen months (Quarter 1 -- Quarter 6). This demonstrates the
benefit of continued development of pipeline analysis software. The fraction of
all host stars with multiple candidates has grown from 17% to 20%, and the
paucity of short-period giant planets in multiple systems is still evident. The
progression toward smaller planets at longer orbital periods with each new
catalog release suggests that Earth-size planets in the Habitable Zone are
forthcoming if, indeed, such planets are abundant.Comment: Submitted to ApJS. Machine-readable tables are available at
http://kepler.nasa.gov, http://archive.stsci.edu/kepler/results.html, and the
NASA Exoplanet Archiv
Planet Occurrence within 0.25 AU of Solar-type Stars from Kepler
We report the distribution of planets as a function of planet radius (R_p),
orbital period (P), and stellar effective temperature (Teff) for P < 50 day
orbits around GK stars. These results are based on the 1,235 planets (formally
"planet candidates") from the Kepler mission that include a nearly complete set
of detected planets as small as 2 Earth radii (Re). For each of the 156,000
target stars we assess the detectability of planets as a function of R_p and P.
We also correct for the geometric probability of transit, R*/a. We consider
first stars within the "solar subset" having Teff = 4100-6100 K, logg =
4.0-4.9, and Kepler magnitude Kp < 15 mag. We include only those stars having
noise low enough to permit detection of planets down to 2 Re. We count planets
in small domains of R_p and P and divide by the included target stars to
calculate planet occurrence in each domain. Occurrence of planets varies by
more than three orders of magnitude and increases substantially down to the
smallest radius (2 Re) and out to the longest orbital period (50 days, ~0.25
AU) in our study. For P < 50 days, the radius distribution is given by a power
law, df/dlogR= k R^\alpha. This rapid increase in planet occurrence with
decreasing planet size agrees with core-accretion, but disagrees with
population synthesis models. We fit occurrence as a function of P to a power
law model with an exponential cutoff below a critical period P_0. For smaller
planets, P_0 has larger values, suggesting that the "parking distance" for
migrating planets moves outward with decreasing planet size. We also measured
planet occurrence over Teff = 3600-7100 K, spanning M0 to F2 dwarfs. The
occurrence of 2-4 Re planets in the Kepler field increases with decreasing
Teff, making these small planets seven times more abundant around cool stars
than the hottest stars in our sample. [abridged]Comment: Submitted to ApJ, 22 pages, 10 figure
KEPLER's First Rocky Planet: Kepler-10b
NASA's Kepler Mission uses transit photometry to determine the frequency of
earth-size planets in or near the habitable zone of Sun-like stars. The mission
reached a milestone toward meeting that goal: the discovery of its first rocky
planet, Kepler-10b. Two distinct sets of transit events were detected: 1) a 152
+/- 4 ppm dimming lasting 1.811 +/- 0.024 hours with ephemeris
T[BJD]=2454964.57375+N*0.837495 days and 2) a 376 +/- 9 ppm dimming lasting
6.86 +/- 0.07 hours with ephemeris T[BJD]=2454971.6761+N*45.29485 days.
Statistical tests on the photometric and pixel flux time series established the
viability of the planet candidates triggering ground-based follow-up
observations. Forty precision Doppler measurements were used to confirm that
the short-period transit event is due to a planetary companion. The parent star
is bright enough for asteroseismic analysis. Photometry was collected at
1-minute cadence for >4 months from which we detected 19 distinct pulsation
frequencies. Modeling the frequencies resulted in precise knowledge of the
fundamental stellar properties. Kepler-10 is a relatively old (11.9 +/- 4.5
Gyr) but otherwise Sun-like Main Sequence star with Teff=5627 +/- 44 K,
Mstar=0.895 +/- 0.060 Msun, and Rstar=1.056 +/- 0.021 Rsun. Physical models
simultaneously fit to the transit light curves and the precision Doppler
measurements yielded tight constraints on the properties of Kepler-10b that
speak to its rocky composition: Mpl=4.56 +/- 1.29 Mearth, Rpl=1.416 +/- 0.036
Rearth, and density=8.8 +/- 2.9 gcc. Kepler-10b is the smallest transiting
exoplanet discovered to date.Comment: Accepted, Astrophysical Journal, November 25, 2010; Eexpected
publication date: February 20, 201
Robust estimation of bacterial cell count from optical density
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data