X-ray Crystal Structure and Specificity of the Plasmodium falciparum Malaria Aminopeptidase PfM18AAP

The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.     

The intracellular distribution of adenylate kinase in the leaves of spinach,wheat and barley

Of the total adenylate-kinase activity in 10-d-old barley and wheat leaves, 40–50% is localised in the chloroplasts, while in mature spinach leaves 50–70% of the enzyme is chloroplastic. The extra-chloroplastic adenylate-kinase activity is associated with the mitochondria, very little, if any, is freely soluble in the cytoplasm. The adenylate pool of the cytoplasm could have access to adenylate-kinase activity in the intermitochondrial space because of the free permeation of adenylates across the outer mitochondrial membrane. Thus the adenylate pool of the cytoplasm could be subject to adenylate-kinase equilibrium. The mitochondrial adenylate kinase appeared to the localised exclusively in the intermembrane space.     

A Quantitative Diffuse Reflectance Imaging (QDRI) System for Comprehensive Surveillance of the Morphological Landscape in Breast Tumor Margins

In an ongoing effort to address the clear clinical unmet needs surrounding breast conserving surgery (BCS), our group has developed a next-generation multiplexed optical-fiber-based tool to assess breast tumor margin status during initial surgeries. Specifically detailed in this work is the performance and clinical validation of a research-grade intra-operative tool for margin assessment based on diffuse optical spectroscopy. Previous work published by our group has illustrated the proof-of-concept generations of this device; here we incorporate a highly optimized quantitative diffuse reflectance imaging (QDRI) system utilizing a wide-field (imaging area = 17cm²) 49-channel multiplexed fiber optic probe, a custom raster-scanning imaging platform, a custom dual-channel white LED source, and an astronomy grade imaging CCD and spectrograph. The system signal to noise ratio (SNR) was found to be greater than 40dB for all channels. Optical property estimation error was found to be less than 10%, on average, over a wide range of absorption (μ_a = 0–8.9cm^-1) and scattering (μ_s’ = 7.0–9.7cm^-1) coefficients. Very low inter-channel and CCD crosstalk was observed (2% max) when used on turbid media (including breast tissue). A raster-scanning mechanism was developed to achieve sub-pixel resolution and was found to be optimally performed at an upsample factor of 8, affording 0.75mm spatially resolved diffuse reflectance images (λ = 450–600nm) of an entire margin (area = 17cm²) in 13.8 minutes (1.23cm²/min). Moreover, controlled pressure application at the probe-tissue interface afforded by the imaging platform reduces repeated scan variability, providing <1% variation across repeated scans of clinical specimens. We demonstrate the clinical utility of this device through a pilot 20-patient study of high-resolution optical parameter maps of the ratio of the β-carotene concentration to the reduced scattering coefficient. An empirical cumulative distribution function (eCDF) analysis is used to reduce optical property maps to quantitative distributions representing the morphological landscape of breast tumor margins. The optimizations presented in this work provide an avenue to rapidly survey large tissue areas on intra-operative time scales with improved sensitivity to regions of focal disease that may otherwise be overlooked.     

Large,Omega-3 Rich,Pelagic Diatoms under Arctic Sea Ice: Sources and Implications for Food Webs

Pelagic primary production in Arctic seas has traditionally been viewed as biologically insignificant until after the ice breakup. There is growing evidence however, that under-ice blooms of pelagic phytoplankton may be a recurrent occurrence. During the springs of 2011 and 2012, we found substantial numbers (201–5713 cells m⁻³) of the large centric diatom (diameter >250 µm) Coscinodiscus centralis under the sea ice in the Canadian Arctic Archipelago near Resolute Bay, Nunavut. The highest numbers of these pelagic diatoms were observed in Barrow Strait. Spatial patterns of fatty acid profiles and stable isotopes indicated two source populations for C. centralis: a western origin with low light conditions and high nutrients, and a northern origin with lower nutrient levels and higher irradiances. Fatty acid analysis revealed that pelagic diatoms had significantly higher levels of polyunsaturated fatty acids (mean ± SD: 50.3±8.9%) compared to ice-associated producers (30.6±10.3%) in our study area. In particular, C. centralis had significantly greater proportions of the long chain omega-3 fatty acid, eicosapentaenoic acid (EPA), than ice algae (24.4±5.1% versus 13.7±5.1%, respectively). Thus, C. centralis represented a significantly higher quality food source for local herbivores than ice algae, although feeding experiments did not show clear evidence of copepod grazing on C. centralis. Our results suggest that C. centralis are able to initiate growth under pack ice in this area and provide further evidence that biological productivity in ice-covered seas may be substantially higher than previously recognized.     

Identification of Biological Markers of Liver X Receptor (LXR) Activation at the Cell Surface of Human Monocytes

Background

Liver X receptor (LXR) α and LXR β (NR1H3 and NR1H2) are oxysterol-activated nuclear receptors involved in the control of major metabolic pathways such as cholesterol homeostasis, lipogenesis, inflammation and innate immunity. Synthetic LXR agonists are currently under development and could find applications in various fields such as cardiovascular diseases, cancer, diabetes and neurodegenerative diseases. The clinical development of LXR agonists requires the identification of biological markers for pharmacodynamic studies. In this context, monocytes represent an attractive target to monitor LXR activation. They are easily accessible cells present in peripheral blood; they express LXR α and β and respond to LXR agonist stimulation in vitro. The aim of our study was to identify cell surface markers of LXR agonists on monocytes. For this, we focused on clusters of differentiation (CD) markers because they are well characterized and accessible cell surface molecules allowing easy immuno-phenotyping.

Methodology/Principal Findings

By using microarray analysis of monocytes treated or not with an LXR agonist in vitro, we selected three CD, i.e. CD82, CD226, CD244 for further analysis by real time PCR and flow cytometry. The three CD were up-regulated by LXR agonist treatment in vitro in a time- and dose- dependent manner and this induction was LXR specific as assessed by a SiRNA or LXR antagonist strategy. By using flow cytometry, we could demonstrate that the expression of these molecules at the cell surface of monocytes was significantly increased after LXR agonist treatment.

Conclusions/Significance

We have identified three new cell surface markers that could be useful to monitor LXR activation. Future studies will be required to confirm the biological and diagnostic significance of the markers.     

Behavioural and Physiological Effects of Finely Balanced Decision-Making in Chickens

In humans, more difficult decisions result in behavioural and physiological changes suggestive of increased arousal, but little is known about the effect of decision difficulty in other species. A difficult decision can have a number of characteristics; we aimed to monitor how finely balanced decisions, compared to unbalanced ones, affected the behaviour and physiology of chickens. An unbalanced decision was one in which the two options were of unequal net value (1 (Q1) vs. 6 (Q6) pieces of sweetcorn with no cost associated with either option); a finely balanced decision was one in which the options were of equal net value (i.e. hens were "indifferent" to both options). To identify hens'' indifference, a titration procedure was used in which a cost (electromagnetic weight on an access door) was applied to the Q6 option, to find the individual point at which hens chose this option approximately equally to Q1 via a non-weighted door. We then compared behavioural and physiological indicators of arousal (head movements, latency to choose, heart-rate variability and surface body temperature) when chickens made decisions that were unbalanced or finely balanced. Significant physiological (heart-rate variability) and behavioural (latency to pen) differences were found between the finely balanced and balanced conditions, but these were likely to be artefacts of the greater time and effort required to push through the weighted doors. No other behavioural and physiological measures were significantly different between the decision categories. We suggest that more information is needed on when best to monitor likely changes in arousal during decision-making and that future studies should consider decisions defined as difficult in other ways.     

Anthropogenic nitrogen sources and relationships to riverine nitrogen export in the northeastern U.S.A.

Human activities have greatly altered the nitrogen (N) cycle, accelerating the rate of N fixation in landscapes and delivery of N to water bodies. To examine relationships between anthropogenic N inputs and riverine N export, we constructed budgets describing N inputs and losses for 16 catchments, which encompass a range of climatic variability and are major drainages to the coast of the North Atlantic Ocean along a latitudinal profile from Maine to Virginia. Using data from the early 1990's, we quantified inputs of N to each catchment from atmospheric deposition, application of nitrogenous fertilizers, biological nitrogen fixation, and import of N in agricultural products (food and feed). We compared these inputs with N losses from the system in riverine export.The importance of the relative sources varies widely by catchment and is related to land use. Net atmospheric deposition was the largest N source (>60%) to the forested basins of northern New England (e.g. Penobscot and Kennebec); net import of N in food was the largest source of N to the more populated regions of southern New England (e.g. Charles & Blackstone); and agricultural inputs were the dominant N sources in the Mid-Atlantic region (e.g. Schuylkill & Potomac). Over the combined area of the catchments, net atmospheric deposition was the largest single source input (31%), followed by net imports of N in food and feed (25%), fixation in agricultural lands (24%), fertilizer use (15%), and fixation in forests (5%). The combined effect of fertilizer use, fixation in crop lands, and animal feed imports makes agriculture the largest overall source of N. Riverine export of N is well correlated with N inputs, but it accounts for only a fraction (25%) of the total N inputs. This work provides an understanding of the sources of N in landscapes, and highlights how human activities impact N cycling in the northeast region.     

Amplification of CRAC current by STIM1 and CRACM1 (Orai1)

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.     

Phenome-Wide Association Studies on a Quantitative Trait: Application to TPMT Enzyme Activity and Thiopurine Therapy in Pharmacogenomics

Phenome-Wide Association Studies (PheWAS) investigate whether genetic polymorphisms associated with a phenotype are also associated with other diagnoses. In this study, we have developed new methods to perform a PheWAS based on ICD-10 codes and biological test results, and to use a quantitative trait as the selection criterion. We tested our approach on thiopurine S-methyltransferase (TPMT) activity in patients treated by thiopurine drugs. We developed 2 aggregation methods for the ICD-10 codes: an ICD-10 hierarchy and a mapping to existing ICD-9-CM based PheWAS codes. Eleven biological test results were also analyzed using discretization algorithms. We applied these methods in patients having a TPMT activity assessment from the clinical data warehouse of a French academic hospital between January 2000 and July 2013. Data after initiation of thiopurine treatment were analyzed and patient groups were compared according to their TPMT activity level. A total of 442 patient records were analyzed representing 10,252 ICD-10 codes and 72,711 biological test results. The results from the ICD-9-CM based PheWAS codes and ICD-10 hierarchy codes were concordant. Cross-validation with the biological test results allowed us to validate the ICD phenotypes. Iron-deficiency anemia and diabetes mellitus were associated with a very high TPMT activity (p = 0.0004 and p = 0.0015, respectively). We describe here an original method to perform PheWAS on a quantitative trait using both ICD-10 diagnosis codes and biological test results to identify associated phenotypes. In the field of pharmacogenomics, PheWAS allow for the identification of new subgroups of patients who require personalized clinical and therapeutic management.     

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.