Cellular Uptake and Efflux of Palbociclib In Vitro in Single Cell and Spheroid Models.

Adequate drug distribution through tumors is essential for treatment to be effective. Palbociclib is a cyclin-dependent kinase 4/6 inhibitor approved for use in patients with hormone receptor positive, human epidermal growth factor receptor 2 negative metastatic breast cancer. It has unusual physicochemical properties, which may significantly influence its distribution in tumor tissue. We studied the penetration and distribution of palbociclib in vitro, including the use of multicellular three-dimensional models and mathematical modeling. MCF-7 and DLD-1 cell lines were grown as single cell suspensions (SCS) and spheroids; palbociclib uptake and efflux were studied using liquid chromatography-tandem mass spectrometry. Intracellular concentrations of palbociclib for MCF-7 SCS (C max 3.22 µM) and spheroids (C max 2.91 µM) were 32- and 29-fold higher and in DLD-1, 13- and 7-fold higher, respectively, than the media concentration (0.1 µM). Total palbociclib uptake was lower in DLD-1 cells than MCF-7 cells in both SCS and spheroids. Both uptake and efflux of palbociclib were slower in spheroids than SCS. These data were used to develop a mathematical model of palbociclib transport that quantifies key parameters determining drug penetration and distribution. The model reproduced qualitatively most features of the experimental data and distinguished between SCS and spheroids, providing additional support for hypotheses derived from the experimental data. Mathematical modeling has the potential for translating in vitro data into clinically relevant estimates of tumor drug concentrations. SIGNIFICANCE STATEMENT: This study explores palbociclib uptake and efflux in single cell suspension and spheroid models of cancer. Large intracellular concentrations of palbociclib are found after drug exposure. The data from this study may aid understanding of the intratumoural pharmacokinetics of palbociclib, which is useful in understanding how drug distributes within tumor tissue and optimizing drug efficacy. Biomathematical modelling has the potential to derive intratumoural drug concentrations from plasma pharmacokinetics in patients.

Evaluation of Scintillator Detection Materials for Application within Airborne Environmental Radiation Monitoring.

In response to the Fukushima Daiichi Nuclear Power Plant accident, there has occurred the unabated growth in the number of airborne platforms developed to perform radiation mapping-each utilising various designs of a low-altitude uncrewed aerial vehicle. Alongside the associated advancements in the airborne system transporting the radiation detection payload, from the earliest radiological analyses performed using gas-filled Geiger-Muller tube detectors, modern radiation detection and mapping platforms are now based near-exclusively on solid-state scintillator detectors. With numerous varieties of such light-emitting crystalline materials now in existence, this combined desk and computational modelling study sought to evaluate the best-available detector material compatible with the requirements for low-altitude autonomous radiation detection, localisation and subsequent high spatial-resolution mapping of both naturally occurring and anthropogenically-derived radionuclides. The ideal geometry of such detector materials is also evaluated. While NaI and CsI (both elementally doped) are (and will likely remain) the mainstays of radiation detection, LaBr3 scintillation detectors were determined to possess not only a greater sensitivity to incident gamma-ray radiation, but also a far superior spectral (energy) resolution over existing and other potentially deployable detector materials. Combined with their current competitive cost, an array of three such composition cylindrical detectors were determined to provide the best means of detecting and discriminating the various incident gamma-rays.

Essential medicines containing ethanol elevate blood acetaldehyde concentrations in neonates.

UNLABELLED: Neonates administered ethanol-containing medicines are potentially at risk of dose-dependent injury through exposure to ethanol and its metabolite, acetaldehyde. Here, we determine blood ethanol and acetaldehyde concentrations in 49 preterm infants (median birth weight = 1190 g) dosed with iron or furosemide, medicines that contain different amounts of ethanol, and in 11 control group infants (median birth weight = 1920 g) who were not on any medications. Median ethanol concentrations in neonates administered iron or furosemide were 0.33 (range = 0-4.92) mg/L, 0.39 (range = 0-72.77) mg/L and in control group infants were 0.15 (range = 0.03-5.4) mg/L. Median acetaldehyde concentrations in neonates administered iron or furosemide were 0.16 (range = 0-8.89) mg/L, 0.21 (range = 0-2.43) mg/L and in control group infants were 0.01 (range = 0-0.14) mg/L. There was no discernible relationship between blood ethanol or acetaldehyde concentrations and time after medication dose. CONCLUSION: Although infants dosed with iron or furosemide had low blood ethanol concentrations, blood acetaldehyde concentrations were consistent with moderate alcohol exposure. The data suggest the need to account for the effects of acetaldehyde in the benefit-risk analysis of administering ethanol-containing medicines to neonates. WHAT IS KNOWN: • Neonates are commonly treated with ethanol-containing medicines, such as iron and furosemide. • However, there is no data on whether this leads to appreciable increases in blood concentrations of ethanol or its metabolite, acetaldehyde. What is New: • In this study, we find low blood ethanol concentrations in neonates administered iron and/or furosemide but markedly elevated blood acetaldehyde concentrations in some infants receiving these medicines. • Our data suggest that ethanol in drugs may cause elevation of blood acetaldehyde, a potentially toxic metabolite.

Fungal endophytes enhance wheat heat and drought tolerance in terms of grain yield and second-generation seed viability.

AIMS: We evaluated the impact of fungal endophyte symbiosis on the growth, ecophysiological and reproductive success of wheat exposed to heat and drought. METHODS AND RESULTS: The resistance of pot-grown wheat to heat or drought stress was measured by quantifying efficiency of photosystem II (Fv /Fm), plant height, average seed weight (ASW), total seed weight (TSW), water-use efficiency (WUE) as well as time to 50% germination and percentage germination of second-generation seeds produced under heat stress, drought stress or well-watered conditions. The endophytic fungi tested increased wheat tolerance for drought and heat. Endophyte SMCD 2206 was the most beneficial, followed by SMCD 2210 and 2215. Surprisingly, second-generation seeds produced by drought-stressed wheat colonized by SMCD 2206, 2210 or 2215 had decreased WUE relative to those produced by endophyte-free, drought-stressed plants. However, these seeds germinated more rapidly than those produced by endophyte-free, stressed parental plants. CONCLUSIONS: The tested consortium of endophytes has the potential to improve wheat adaptation to heat and drought. SIGNIFICANCE AND IMPACT OF THE STUDY: The capacity of endophytes to increase wheat tolerance for abiotic stress and to improved germination in endophyte-free second-generation seeds arising from stressed plants could be applicable to agriculture. The mechanisms by which intergenerational endophyte-mediated affects occurs warrant further research.

Multiphase modelling of vascular tumour growth in two spatial dimensions.

In this paper we present a continuum mathematical model of vascular tumour growth which is based on a multiphase framework in which the tissue is decomposed into four distinct phases and the principles of conservation of mass and momentum are applied to the normal/healthy cells, tumour cells, blood vessels and extracellular material. The inclusion of a diffusible nutrient, supplied by the blood vessels, allows the vasculature to have a nonlocal influence on the other phases. Two-dimensional computational simulations are carried out on unstructured, triangular meshes to allow a natural treatment of irregular geometries, and the tumour boundary is captured as a diffuse interface on this mesh, thereby obviating the need to explicitly track the (potentially highly irregular and ill-defined) tumour boundary. A hybrid finite volume/finite element algorithm is used to discretise the continuum model: the application of a conservative, upwind, finite volume scheme to the hyperbolic mass balance equations and a finite element scheme with a stable element pair to the generalised Stokes equations derived from momentum balance, leads to a robust algorithm which does not use any form of artificial stabilisation. The use of a matrix-free Newton iteration with a finite element scheme for the nutrient reaction-diffusion equations allows full nonlinearity in the source terms of the mathematical model. Numerical simulations reveal that this four-phase model reproduces the characteristic pattern of tumour growth in which a necrotic core forms behind an expanding rim of well-vascularised proliferating tumour cells. The simulations consistently predict linear tumour growth rates. The dependence of both the speed with which the tumour grows and the irregularity of the invading tumour front on the model parameters is investigated.

Solar eclipses of Phobos and Deimos observed from the surface of Mars.

The small martian satellites Phobos and Deimos orbit in synchronous rotation with inclinations of only 0.01 degrees and 0.92 degrees , respectively, relative to the planet's equatorial plane. Thus, an observer at near-equatorial latitudes on Mars could occasionally observe solar eclipses by these satellites (see ref. 1, for example). Because the apparent angular diameter of the satellites is much smaller than that of the Sun, however, such events are more appropriately referred to as transits. Transit data can be used for correcting and refining the orbital ephemerides of the moons. For example, Phobos is known to exhibit a secular acceleration that is caused by tidal dissipation within Mars. Long-term, accurate measurements are needed to refine the magnitude and origin of this dissipation within the martian interior as well as to refine the predicted orbital evolution of both satellites. Here we present observations of six transits of Phobos and Deimos across the solar disk from cameras on Mars aboard the Mars Exploration Rovers Spirit and Opportunity. These are the first direct imaging observations of satellites transiting the Sun from the surface of another planet.

On target with a new mechanism for the regulation of protein phosphorylation.

There is overwhelming evidence that the reversible phosphorylation of proteins regulates most aspects of cell life. However, the broad specificities displayed by many protein phosphatases and kinases in vitro dictates that their activities be strictly regulated in vivo. Recent evidence indicates that a novel class of proteins, known as targetting subunits, specifies the location, catalytic and regulatory properties of protein phosphatases and kinases, and thereby plays a key role in ensuring the fidelity of protein phosphorylation.

Upper ocean oxygenation, evolution of RuBisCO and the Phanerozoic succession of phytoplankton.

Evidence is compiled to demonstrate a redox scale within Earth's photosynthesisers that correlates the specificity of their RuBisCO with organismal metabolic tolerance to anoxia, and ecological selection by dissolved O2/CO2 and nutrients. The Form 1B RuBisCO found in the chlorophyte green algae, has a poor selectivity between the two dissolved substrates, O2 and CO2, at the active site. This enzyme appears adapted to lower O2/CO2 ratios, or more "anoxic" conditions and therefore requires additional energetic or nutrient investment in a carbon concentrating mechanism (CCM) to boost the intracellular CO2/O2 ratio and maintain competitive carboxylation rates under increasingly high O2/CO2 conditions in the environment. By contrast the coccolithophores and diatoms evolved containing the more selective Rhodophyte Form 1D RuBisCO, better adapted to a higher O2/CO2 ratio, or more oxic conditions. This Form 1D RuBisCO requires lesser energetic or nutrient investment in a CCM to attain high carboxylation rates under environmentally high O2/CO2 ratios. Such a physiological relationship may underpin the succession of phytoplankton in the Phanerozoic oceans: the coccolithophores and diatoms took over the oceanic realm from the incumbent cyanobacteria and green algae when the upper ocean became persistently oxygenated, alkaline and more oligotrophic. The facultatively anaerobic green algae, able to tolerate the anoxic conditions of the water column and a periodically inundated soil, were better poised to adapt to the fluctuating anoxia associated with periods of submergence and emergence and transition onto the land. The induction of a CCM may exert a natural limit to the improvement of RuBisCO efficiency over Earth history. Rubisco specificity appears to adapt on the timescale of ∼100 Myrs. So persistent elevation of CO2/O2 ratios in the intracellular environment around the enzyme, may induce a relaxation in RuBisCO selectivity for CO2 relative to O2. The most efficient RuBisCO for net carboxylation is likely to be found in CCM-lacking algae that have been exposed to hyperoxic conditions for at least 100 Myrs, such as intertidal brown seaweeds.

Effective equations governing an active poroelastic medium.

In this work, we consider the spatial homogenization of a coupled transport and fluid-structure interaction model, to the end of deriving a system of effective equations describing the flow, elastic deformation and transport in an active poroelastic medium. The 'active' nature of the material results from a morphoelastic response to a chemical stimulant, in which the growth time scale is strongly separated from other elastic time scales. The resulting effective model is broadly relevant to the study of biological tissue growth, geophysical flows (e.g. swelling in coals and clays) and a wide range of industrial applications (e.g. absorbant hygiene products). The key contribution of this work is the derivation of a system of homogenized partial differential equations describing macroscale growth, coupled to transport of solute, that explicitly incorporates details of the structure and dynamics of the microscopic system, and, moreover, admits finite growth and deformation at the pore scale. The resulting macroscale model comprises a Biot-type system, augmented with additional terms pertaining to growth, coupled to an advection-reaction-diffusion equation. The resultant system of effective equations is then compared with other recent models under a selection of appropriate simplifying asymptotic limits.

Drug delivery in a tumour cord model: a computational simulation.

The tumour vasculature and microenvironment is complex and heterogeneous, contributing to reduced delivery of cancer drugs to the tumour. We have developed an in silico model of drug transport in a tumour cord to explore the effect of different drug regimes over a 72 h period and how changes in pharmacokinetic parameters affect tumour exposure to the cytotoxic drug doxorubicin. We used the model to describe the radial and axial distribution of drug in the tumour cord as a function of changes in the transport rate across the cell membrane, blood vessel and intercellular permeability, flow rate, and the binding and unbinding ratio of drug within the cancer cells. We explored how changes in these parameters may affect cellular exposure to drug. The model demonstrates the extent to which distance from the supplying vessel influences drug levels and the effect of dosing schedule in relation to saturation of drug-binding sites. It also shows the likely impact on drug distribution of the aberrant vasculature seen within tumours. The model can be adapted for other drugs and extended to include other parameters. The analysis confirms that computational models can play a role in understanding novel cancer therapies to optimize drug administration and delivery.

The impact of electrode resistance on the biogalvanic characterisation technique.

Measurement of a tissue-specific electrical resistance may offer a discriminatory metric for evaluation of tissue health during cancer surgery. With a move toward minimally-invasive procedures, applicable contact sensing modalities must be scalable, fast and robust. A passive resistance characterisation method utilising a biogalvanic cell as an intrinsic power source has been proposed as a potentially suitable solution. Previous work has evaluated this system with results showing effective discrimination of tissue type and damage (through electroporation). However, aspects of the biogalvanic cell have been found to influence the characterisation performance, and are not currently accounted for within the system model. In particular, the electrode and salt-bridge resistance are not independently determined, leading to over-predictions of tissue resistivity. This paper describes a more comprehensive model and characterisation scheme, with electrode parameters and salt-bridge resistivity being evaluated independently. In a generalised form, the presented model illustrates how the relative resistive contributions from the electrodes and medium relate to the existing characterisation method efficacy. We also describe experiments with physiologically relevant salt solutions (1.71, 17.1, 154 mM), used for validation and comparison. The presented model shows improved performance over the current biogalvanic measurement technique at the median conductivity. Both the proposed and extant system models become unable to predict conductivity accurately at high conductivity due to the dominance of the electrodes. The characterisation techniques have also been applied to data collected on freshly excised human colon tissue (healthy and cancerous). The findings suggest that the resistance of the cell under the test conditions is electrode dominated, leading to erroneous tissue resistance determination. Measurement optimisation strategies and the surgical applicability of the biogalvanic technique are discussed in light of these findings.

Differential feeding-related regulation of ubiquitin and calbindin9kDa in rat duodenum.

Analyses of the calcium-binding protein, calbindin9kDa, purified to apparent homogeneity (SDS-PAGE) from rat duodenum, revealed variable contamination by two other 9 kDa proteins (up to 0.2 mol equivalent each) which were identified as ubiquitin and its C-terminal variant, des-Gly-Gly-ubiquitin. We found that the co-purification of these proteins did not reflect a tight molecular interaction but instead their unexpectedly similar physical characteristics in nondenaturing conditions. Like calbindin9kDa, free ubiquitin was abundant (1% and 0.4% of soluble protein, respectively) in duodenum mucosa of 7-8-week-old rats and its concentration varied daily and with feeding status. In rats fed from midnight to 8.30 a.m., the ubiquitin concentration was specifically higher at 10 pm than at 10 a.m. (11.2 +/- 0.7 and 7.7 +/- 0.8 nmol per g wet weight, respectively, P < 0.02), whereas calbindin9kDa tended towards an opposite variation (18.0 +/- 1.9 and 21.8 +/- 1.7 nmol per g, respectively). Based on its unusually high abundance and novel feeding-related variations, ubiquitin must have an important functional role in the rat duodenum which is distinctly regulated from the calcium transport-associated role of calbindin9kDa.

Partial structure and hormonal regulation of rabbit liver inhibitor-1; distribution of inhibitor-1 and inhibitor-2 in rabbit and rat tissues.

Inhibitor-1 purified from rabbit liver could not be distinguished from the skeletal muscle protein by chromatographic, electrophoretic and immunological criteria. Amino acid sequences comprising 68% of rabbit liver inhibitor-1 were identical to the skeletal muscle protein indicating that they are products of a single gene. Total inhibitor-1 activity in heat-treated rabbit liver extracts was similar to that in skeletal muscle extracts, and the phosphorylation state of inhibitor-1 increased from 14% to 42% in rabbit liver in vivo after an intravenous injection of glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-1 recognised a single major protein of identical electrophoretic mobility (26 kDa) in each rabbit tissue examined (skeletal muscle, liver, brain, heart, kidney, uterus and adipose). The antibodies also recognised a single major (30 kDa) protein in the same rat tissues, except liver. The results show that while there are interspecies differences in apparent molecular mass, inhibitor-1 is likely to be the same gene product in each mammalian tissue. Inhibitor-1 was not detected in rat liver, either by activity measurements or immunoblotting, irrespective of the age, sex or strain of the animals. Immunoblotting also failed to detect inhibitor-1 in mouse liver, although it was present in guinea pig, porcine and sheep liver. The absence of inhibitor-1 in rat liver indicates that phosphorylation of this protein cannot underlie the increased phosphorylation of hydroxymethylglutaryl-CoA reductase observed after stimulation by glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-2 recognised a 31 kDa protein in each rabbit tissue, and a 33 kDa protein in all rat tissues including liver. The results suggest that inhibitor-2 is the same gene product in each mammalian tissue.

Lysozyme and alpha-lactalbumin from the milk of a marsupial, the common brush-tailed possum (Trichosurus vulpecula).

Lysozyme and alpha-lactalbumin have been identified using N-terminal sequence analysis of whey proteins from the common brush-tailed possum, Trichosurus vulpecula after separation by two-dimensional denaturing electrophoresis. Both proteins were purified from pooled possum milk using ion exchange chromatography and gave mass values of 14,896 and 13,985 Da respectively by MALDI-TOF mass spectrometry. Clones containing the full coding sequences of the genes for both proteins were isolated from a possum mammary cDNA library and the DNA sequence of the coding region determined. The inferred protein sequences were used in phylogenetic analysis of both protein classes. These showed that the T. vulpecula alpha-lactalbumin, along with other marsupial alpha-lactalbumins, formed a family distinct from the eutherian alpha-lactalbumins and the alpha-lactalbumin of a monotreme, the platypus, consistent with the separate evolution of the marsupials. By contrast the T. vulpecula lysozyme was shown to be similar to the ruminant stomach lysozymes and primate lysozymes and quite distinct from the Ca2+-binding lysozymes found in the milk of the echidna and horse.

Hemoglobin Atlanta or alpha 2 beta 2 75 Leu-Pro (E19): an unstable variant found in several members of a Caucasian family.

Hemoglobin Atlanta, alpha 2 beta 2 75 Leu-Pro (E19), has been found in several members of three generations of a Caucasian family living in metropolitan Atlanta. The abnormal hemoglobin is one of the nine unstable variants in which either a leucyl or an alanyl residue is replaced by a prolyl residue. These substitutions have been observed in the B, E, F, and G helixes of the beta chain and in the H helix of the alpha-chain. Hemoglobin Atlanta heterozygotes are mildly affected by the presence of this unstable hemoglobin.

Controlled study of haloperidol, pimozide and placebo for the treatment of Gilles de la Tourette's syndrome.

The results of this controlled study of the treatment of 57 patients with Gilles de la Tourette's syndrome suggested that both haloperidol and pimozide were more effective than placebo, but that haloperidol was slightly more effective than pimozide. Adverse effects occurred more frequently with haloperidol vs placebo than with pimozide vs placebo, but the frequency was not significantly different for haloperidol compared with pimozide. Clinically significant cardiac effects did not occur at a maximum dosage of 0.3 mg/kg or 20 mg/d for pimozide and 10 mg/d for haloperidol. However, the QTc interval was prolonged during pimozide treatment compared with that during haloperidol treatment, although the values for both medications were not in an abnormal range.

Intracellular cytokines in the acute response to highly active antiretroviral therapy.

OBJECTIVES: Successful highly active antiretroviral therapy (HAART) is usually associated with a rapid decline in HIV plasma RNA levels and a gradual increase in CD4 T cells. We examined whether changes in cytokine production and profile precede other immunological changes and whether these might occur in temporal association with plasma HIV RNA changes. DESIGN AND METHODS: Eleven HIV-1-infected patients were enrolled into a prospective cohort study; eight patients were naive to antiretroviral therapy. Blood samples were collected pre-therapy (week 0) and at 1, 2, and 3 weeks post-initiation of therapy. RESULTS: All 11 patients enrolled remained on triple HAART for 1 week, eight for 2 weeks, and six for > or = 3 weeks. When compared to week 0, these patients had a > or = 2-log10 decline in HIV plasma RNA levels and/or a decline to < or = 400 copies/ml by week 3 of therapy (p = 0.004). The numbers and percentages of CD4 and CD8 T cells, and the percentage of naive, memory, and activated T cells did not change significantly between weeks 0 and 1 or 0 and 3. Of all the immune parameters examined only: the percentage of CD4 T cells spontaneously producing tumor necrosis factor (TNF)-alpha (median, 2.4 versus 0.5% P = 0.025); the percentage of CD8 T cells spontaneously producing TNF-alpha (median, 0.6 versus 0.2% P = 0.037); and the percentage of CD3 T cells spontaneously producing interleukin-4 (median, 1.8 versus 0.8% P = 0.004) changed significantly between weeks 0 and 3. CONCLUSIONS: In these patients, decreases in the percentage of T cells spontaneously producing TNF-alpha or interleukin-4 preceded changes in CD4 T cells. If confirmed by others, these observations may be useful as early predictors of response to and early failure of HAART.

Use of ultrasonography-assisted liposuction for the treatment of human immunodeficiency virus-associated enlargement of the dorsocervical fat pad.

Enlargement of the dorsocervical fat pad (i.e., "buffalo hump") is one manifestation of the lipodystrophy syndrome associated with human immunodeficiency virus. We report our experience with the use of ultrasonography-assisted liposuction in a cohort of 10 patients with this complication.

Radial and vertebral bone density in white and black women: evidence for racial differences in premenopausal bone homeostasis.

The reasons for a different incidence of osteoporotic fractures in white and black women are unknown. Previous racial comparisons of bone mass have been limited by racial differences in body weight and socioeconomic, health, and nutritional status. This cross-sectional study examined bone density in 105 black and 114 white healthy nonobese women, 24-65 yr old, using dual photon absorptiometry of the lumbar spine and single photon absorptiometry of the distal radius. Bone density at both sites was higher in blacks at all ages than in whites. When adjusted for age and body mass index, mean bone density was 6.5% higher in blacks at both spine and radius (P less than 0.0001). The cross-sectional rate of decline of vertebral bone density was similar between races; however, radial density increased 3.8%/decade (P = 0.03) in premenopausal blacks under age 46 yr, while it declined 3.2%/decade (P = 0.09) in premenopausal whites. The racial difference in slopes in these premenopausal women is significant (P = 0.002). These findings suggest that attainment of higher peak bone mass and delayed onset of bone loss contribute to the lower incidence of osteoporotic fractures in black women.