Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed

The temporal reliability of serum estrogens, progesterone, gonadotropins, SHBG and urinary estrogen and progesterone metabolites in premenopausal women

BACKGROUND: There is little existing research to guide researchers in estimating the minimum number of measurement occasions required to obtain reliable estimates of serum estrogens, progesterone, gonadotropins, sex hormone-binding globulin (SHBG), and urinary estrogen and progesterone metabolites in premenopausal women. METHODS: Using data from a longitudinal study of 34 women with a mean age of 42.3 years (SD = 2.6), we calculated the minimum number of measurement occasions required to obtain reliable estimates of 12 analytes (8 in blood, 4 in urine). Five samples were obtained over 1 year: at baseline, and after 1, 3, 6, and 12 months. We also calculated the percent of true variance accounted for by a single measurement and intraclass correlation coefficients (ICC) between measurement occasions. RESULTS: Only 2 of the 12 analytes we examined, SHBG and estrone sulfate (E(1)S), could be adequately estimated by a single measurement using a minimum reliability standard of having the potential to account for 64% of true variance. Other analytes required from 2 to 12 occasions to account for 81% of the true variance, and 2 to 5 occasions to account for 64% of true variance. ICCs ranged from 0.33 for estradiol (E(2)) to 0.88 for SHBG. Percent of true variance accounted for by single measurements ranged from 29% for luteinizing hormone (LH) to 92% for SHBG. CONCLUSIONS: Experimental designs that take the natural variability of these analytes into account by obtaining measurements on a sufficient number of occasions will be rewarded with increased power and accuracy

Hypercalcemia in a patient with disseminated paracoccidioidomycosis: a case report

<p>Abstract</p> <p>Introduction</p> <p>Hypercalcemia is well described in various granulomatous disorders, such as sarcoidosis, tuberculosis, berylliosis, leprosy and fungal infections. However, the association of <it>Paracoccidioides brasiliensis </it>and hypercalcemia is rare: to the best of our knowledge, only two cases have previously been reported, and neither had a clear documentation of the etiology of the hypercalcemia.</p> <p>Case presentation</p> <p>We report the case of a 22-year-old man in whom disseminated infection with paracoccidioidomycosis was associated with hypercalcemia. The patient had a high normal serum level of 1,25-dihydroxyvitamin D and a suppressed parathyroid hormone value, an indication that the hypercalcemia was not mediated by parathyroid hormone and might be associated with 1,25-dihydroxyvitamin D.</p> <p>Conclusion</p> <p>The episode resolved readily with administration of corticosteroids, an outcome suggesting that this is an effective treatment of hypercalcemia of this origin. On follow-up, while receiving antifungal therapy for <it>P. brasiliensis </it>the patient's calcium values remained normal.</p

Meta-All: a system for managing metabolic pathway information

BACKGROUND: Many attempts are being made to understand biological subjects at a systems level. A major resource for these approaches are biological databases, storing manifold information about DNA, RNA and protein sequences including their functional and structural motifs, molecular markers, mRNA expression levels, metabolite concentrations, protein-protein interactions, phenotypic traits or taxonomic relationships. The use of these databases is often hampered by the fact that they are designed for special application areas and thus lack universality. Databases on metabolic pathways, which provide an increasingly important foundation for many analyses of biochemical processes at a systems level, are no exception from the rule. Data stored in central databases such as KEGG, BRENDA or SABIO-RK is often limited to read-only access. If experimentalists want to store their own data, possibly still under investigation, there are two possibilities. They can either develop their own information system for managing that own data, which is very time-consuming and costly, or they can try to store their data in existing systems, which is often restricted. Hence, an out-of-the-box information system for managing metabolic pathway data is needed. RESULTS: We have designed META-ALL, an information system that allows the management of metabolic pathways, including reaction kinetics, detailed locations, environmental factors and taxonomic information. Data can be stored together with quality tags and in different parallel versions. META-ALL uses Oracle DBMS and Oracle Application Express. We provide the META-ALL information system for download and use. In this paper, we describe the database structure and give information about the tools for submitting and accessing the data. As a first application of META-ALL, we show how the information contained in a detailed kinetic model can be stored and accessed. CONCLUSION: META-ALL is a system for managing information about metabolic pathways. It facilitates the handling of pathway-related data and is designed to help biochemists and molecular biologists in their daily research. It is available on the Web at and can be downloaded free of charge and installed locally

Gratefully received, gratefully repaid:The role of perceived fairness in cooperative interactions

It is well documented that people would remunerate fair behaviours and penalize unfair behaviours. It is argued that individuals' reactions following the receipt of a gift depend on the perceived intentions of the donors. Fair intentions should prompt positive affect, like gratitude, triggering cooperative behaviours; while intended unfairness should trigger negative affect, like anger, fostering anti-social actions. It is, however, contended that when people lack information to infer others' intention they may use 'normative' beliefs about fairness - what a typical fair individual 'should' do in these circumstances - to guide their behaviour. In this experiment we examined this assertion. We had 122 participants play a one-shot, double-anonymous game with half playing as potential helpers (P1s) and half as recipients (P2s). Whether a participant was a P1 or P2 was chance-determined and all participants knew this. P1s decided whether to help P2s and whether to make their help unconditional (no repayment needed) or conditional (full or 'taxed' repayment). P2s decided whether to accept the offer and whatever conditions attached but were blind to the list of helping options available to P1s. We anticipated that recipients would refer to the 'injunctive norm' that 'fair people should help "for free" when it is only by chance that they are in a position to help'. Therefore, without knowing P1s' different helping options, unconditional offers should be rated by recipients as fairer than conditional offers, and this should be linked to greater gratitude with greater gratitude linked to greater reciprocation. Path analyses confirmed this serial mediation. The results showed that recipients of unconditional offers, compared to conditional ones, interpreted the helpers' motives as more helpful, experienced greater gratitude and were more eager to reciprocate. The behavioural data further revealed that, when given a latter option to default, 38% of recipients of conditional offers did so

A generic algorithm for layout of biological networks

BackgroundBiological networks are widely used to represent processes in biological systems and to capture interactions and dependencies between biological entities. Their size and complexity is steadily increasing due to the ongoing growth of knowledge in the life sciences. To aid understanding of biological networks several algorithms for laying out and graphically representing networks and network analysis results have been developed. However, current algorithms are specialized to particular layout styles and therefore different algorithms are required for each kind of network and/or style of layout. This increases implementation effort and means that new algorithms must be developed for new layout styles. Furthermore, additional effort is necessary to compose different layout conventions in the same diagram. Also the user cannot usually customize the placement of nodes to tailor the layout to their particular need or task and there is little support for interactive network exploration.ResultsWe present a novel algorithm to visualize different biological networks and network analysis results in meaningful ways depending on network types and analysis outcome. Our method is based on constrained graph layout and we demonstrate how it can handle the drawing conventions used in biological networks.ConclusionThe presented algorithm offers the ability to produce many of the fundamental popular drawing styles while allowing the exibility of constraints to further tailor these layouts.publishe

Arabidopsis thaliana encodes a bacterial-type heterodimeric isopropylmalate isomerase involved in both Leu biosynthesis and the Met chain elongation pathway of glucosinolate formation

The last steps of the Leu biosynthetic pathway and the Met chain elongation cycle for glucosinolate formation share identical reaction types suggesting a close evolutionary relationship of these pathways. Both pathways involve the condensation of acetyl-CoA and a 2-oxo acid, isomerization of the resulting 2-malate derivative to form a 3-malate derivative, the oxidation-decarboxylation of the 3-malate derivative to give an elongated 2-oxo acid, and transamination to generate the corresponding amino acid. We have now analyzed the genes encoding the isomerization reaction, the second step of this sequence, in Arabidopsis thaliana. One gene encodes the large subunit and three encode small subunits of this enzyme, referred to as isopropylmalate isomerase (IPMI) with respect to the Leu pathway. Metabolic profiling of large subunit mutants revealed accumulation of intermediates of both Leu biosynthesis and Met chain elongation, and an altered composition of aliphatic glucosinolates demonstrating the function of this gene in both pathways. In contrast, the small subunits appear to be specialized to either Leu biosynthesis or Met chain elongation. Green fluorescent protein tagging experiments confirms the import of one of the IPMI small subunits into the chloroplast, the localization of the Met chain elongation pathway in these organelles. These results suggest the presence of different heterodimeric IPMIs in Arabidopsis chloroplasts with distinct substrate specificities for Leu or glucosinolate metabolism determined by the nature of the different small subunit

Short-Term Compassion Training Increases Prosocial Behavior in a Newly Developed Prosocial Game

Compassion has been suggested to be a strong motivator for prosocial behavior. While research has demonstrated that compassion training has positive effects on mood and health, we do not know whether it also leads to increases in prosocial behavior. We addressed this question in two experiments. In Experiment 1, we introduce a new prosocial game, the Zurich Prosocial Game (ZPG), which allows for repeated, ecologically valid assessment of prosocial behavior and is sensitive to the influence of reciprocity, helping cost, and distress cues on helping behavior. Experiment 2 shows that helping behavior in the ZPG increased in participants who had received short-term compassion training, but not in participants who had received short-term memory training. Interindividual differences in practice duration were specifically related to changes in the amount of helping under no-reciprocity conditions. Our results provide first evidence for the positive impact of short-term compassion training on prosocial behavior towards strangers in a training-unrelated task

Mapping Connectivity Damage in the Case of Phineas Gage

White matter (WM) mapping of the human brain using neuroimaging techniques has gained considerable interest in the neuroscience community. Using diffusion weighted (DWI) and magnetic resonance imaging (MRI), WM fiber pathways between brain regions may be systematically assessed to make inferences concerning their role in normal brain function, influence on behavior, as well as concerning the consequences of network-level brain damage. In this paper, we investigate the detailed connectomics in a noted example of severe traumatic brain injury (TBI) which has proved important to and controversial in the history of neuroscience. We model the WM damage in the notable case of Phineas P. Gage, in whom a “tamping iron” was accidentally shot through his skull and brain, resulting in profound behavioral changes. The specific effects of this injury on Mr. Gage's WM connectivity have not previously been considered in detail. Using computed tomography (CT) image data of the Gage skull in conjunction with modern anatomical MRI and diffusion imaging data obtained in contemporary right handed male subjects (aged 25–36), we computationally simulate the passage of the iron through the skull on the basis of reported and observed skull fiducial landmarks and assess the extent of cortical gray matter (GM) and WM damage. Specifically, we find that while considerable damage was, indeed, localized to the left frontal cortex, the impact on measures of network connectedness between directly affected and other brain areas was profound, widespread, and a probable contributor to both the reported acute as well as long-term behavioral changes. Yet, while significantly affecting several likely network hubs, damage to Mr. Gage's WM network may not have been more severe than expected from that of a similarly sized “average” brain lesion. These results provide new insight into the remarkable brain injury experienced by this noteworthy patient

Limits on active to sterile neutrino oscillations from disappearance searches in the MINOS, Daya Bay, and bugey-3 experiments

Searches for a light sterile neutrino have been performed independently by the MINOS and the Daya Bay experiments using the muon (anti)neutrino and electron antineutrino disappearance channels, respectively. In this Letter, results from both experiments are combined with those from the Bugey-3 reactor neutrino experiment to constrain oscillations into light sterile neutrinos. The three experiments are sensitive to complementary regions of parameter space, enabling the combined analysis to probe regions allowed by the Liquid Scintillator Neutrino Detector (LSND) and MiniBooNE experiments in a minimally extended four-neutrino flavor framework. Stringent limits on sin^2 2θμe are set over 6 orders of magnitude in the sterile mass-squared splitting Δm^2 41. The sterile-neutrino mixing phase space allowed by the LSND and MiniBooNE experiments is excluded for Δm^2 41 < 0.8 eV^2 at 95% CLs