Enantioseparation of flurbiprofen enantiomers using chiral ionic liquids by liquid‐liquid extraction

Flurbiprofen is a kind of nonsteroidal anti‐inflammatory drug, which has been widely used in clinic for treatment of rheumatoid arthritis and osteoarthritis. It has been reported that S‐flurbiprofen shows good performance on clinic anti‐inflammatory treatment, while R‐enantiomer almost has no pharmacological activities. It has important practical values to obtain optically pure S‐flurbiprofen. In this work, chiral ionic liquids, which have good structural designability and chiral recognize ability, were selected as the extraction selector by the assistance of quantum chemistry calculations. The distribution behaviors of flurbiprofen enantiomers were investigated in the extraction system, which was composed of organic solvent and aqueous phase containing chiral ionic liquid. The results show that maximum enantioselectivity up to 1.20 was attained at pH 2.0, 25°C using 1,2‐dichloroethane as organic solvent, 1‐butyl‐3‐methylimidazole L‐tryptophan ([Bmim][L‐trp]) as chiral selector. The racemic flurbiprofen initial concentration was 0.2 mmol L−1, and [Bmim][L‐trp] concentration was 0.02 mol L−1. Furthermore, the recycle of chiral ionic liquids has been achieved by reverse extraction process of the aqueous phase with chiral selector, which is significant for industrial application of chiral ionic liquids and scale‐up of the extraction process

Fast, automated measurement of nematode swimming (thrashing) without morphometry

Background: The "thrashing assay", in which nematodes are placed in liquid and the frequency of lateral swimming ("thrashing") movements estimated, is a well-established method for measuring motility in the genetic model organism Caenorhabditis elegans as well as in parasitic nematodes. It is used as an index of the effects of drugs, chemicals or mutations on motility and has proved useful in identifying mutants affecting behaviour. However, the method is laborious, subject to experimenter error, and therefore does not permit high-throughput applications. Existing automation methods usually involve analysis of worm shape, but this is computationally demanding and error-prone. Here we present a novel, robust and rapid method of automatically counting the thrashing frequency of worms that avoids morphometry but nonetheless gives a direct measure of thrashing frequency. Our method uses principal components analysis to remove the background, followed by computation of a covariance matrix of the remaining image frames from which the interval between statistically-similar frames is estimated. Results: We tested the performance of our covariance method in measuring thrashing rates of worms using mutations that affect motility and found that it accurately substituted for laborious, manual measurements over a wide range of thrashing rates. The algorithm used also enabled us to determine a dose-dependent inhibition of thrashing frequency by the anthelmintic drug, levamisole, illustrating the suitability of the system for assaying the effects of drugs and chemicals on motility. Furthermore, the algorithm successfully measured the actions of levamisole on a parasitic nematode, Haemonchus contortus, which undergoes complex contorted shapes whilst swimming, without alterations in the code or of any parameters, indicating that it is applicable to different nematode species, including parasitic nematodes. Our method is capable of analyzing a 30 s movie in less than 30 s and can therefore be deployed in rapid screens. Conclusion: We demonstrate that a covariance-based method yields a fast, reliable, automated measurement of C. elegans motility which can replace the far more time-consuming, manual method. The absence of a morphometry step means that the method can be applied to any nematode that swims in liquid and, together with its speed, this simplicity lends itself to deployment in large-scale chemical and genetic screens. </p

Cyclin D1 and mammary carcinoma: new insights from transgenic mouse models

Cyclin D1 is one of the most commonly overexpressed oncogenes in breast cancer, with 45–50% of primary ductal carcinomas overexpressing this oncoprotein. Targeted deletion of the gene encoding cyclin D1 demonstrates an essential role in normal mammary gland development while transgenic studies provide evidence that cyclin D1 is a weak oncogene in mammary epithelium. In a recent exciting development, Yu et al. demonstrate that cyclin D1-deficient mice are resistant to mammary carcinomas induced by c-neu and v-Ha-ras, but not those induced by c-myc or Wnt-1. These findings define a pivotal role for cyclin D1 in a subset of mammary cancers in mice and imply a functional role for cyclin D1 overexpression in human breast cancer

The alpha 7 nicotinic receptor agonist PHA-543613 hydrochloride inhibits <i>Porphyromonas gingivalis</i>-induced expression of interleukin-8 by oral keratinocytes

Objective: The alpha 7 nicotinic receptor (α7nAChR) is expressed by oral keratinocytes. α7nAChR activation mediates anti-inflammatory responses. The objective of this study was to determine if α7nAChR activation inhibited pathogen-induced interleukin-8 (IL-8) expression by oral keratinocytes.&lt;p&gt;&lt;/p&gt; Materials and methods: Periodontal tissue expression of α7nAChR was determined by real-time PCR. OKF6/TERT-2 oral keratinocytes were exposed to &lt;i&gt;Porphyromonas gingivalis&lt;/i&gt; in the presence and absence of a α7nAChR agonist (PHA-543613 hydrochloride) alone or after pre-exposure to a specific α7nAChR antagonist (α-bungarotoxin). Interleukin-8 (IL-8) expression was measured by ELISA and real-time PCR. Phosphorylation of the NF-κB p65 subunit was determined using an NF-κB p65 profiler assay and STAT-3 activation by STAT-3 in-cell ELISA. The release of ACh from oral keratinocytes in response to &lt;i&gt;P. gingivalis&lt;/i&gt; lipopolysaccharide was determined using a GeneBLAzer M3 CHO-K1-blacell reporter assay.&lt;p&gt;&lt;/p&gt; Results: Expression of α7nAChR mRNA was elevated in diseased periodontal tissue. PHA-543613 hydrochloride inhibited &lt;i&gt;P. Gingivalis&lt;/i&gt;-induced expression of IL-8 at the transcriptional level. This effect was abolished when cells were pre-exposed to a specific α7nAChR antagonist, α-bungarotoxin. PHA-543613 hydrochloride downregulated NF-κB signalling through reduced phosphorylation of the NF-κB p65-subunit. In addition, PHA-543613 hydrochloride promoted STAT-3 signalling by maintenance of phosphorylation. Furthermore, oral keratinocytes upregulated ACh release in response to &lt;i&gt;P. Gingivalis&lt;/i&gt; lipopolysaccharide.&lt;p&gt;&lt;/p&gt; Conclusion: These data suggest that α7nAChR plays a role in regulating the innate immune responses of oral keratinocytes.&lt;p&gt;&lt;/p&gt

The role of noise and positive feedback in the onset of autosomal dominant diseases

<p>Abstract</p> <p>Background</p> <p>Autosomal dominant (AD) diseases result when a single mutant or non-functioning gene is present on an autosomal chromosome. These diseases often do not emerge at birth. There are presently two prevailing theories explaining the expression of AD diseases. One explanation originates from the Knudson two-hit theory of hereditary cancers, where loss of heterozygosity or occurrence of somatic mutations impairs the function of the wild-type copy. While these somatic second hits may be sufficient for stable disease states, it is often difficult to determine if their occurrence necessarily marks the initiation of disease progression. A more direct consequence of a heterozygous genetic background is haploinsufficiency, referring to a lack of sufficient gene function due to reduced wild-type gene copy number; however, haploinsufficiency can involve a variety of additional mechanisms, such as noise in gene expression or protein levels, injury and second hit mutations in other genes. In this study, we explore the possible contribution to the onset of autosomal dominant diseases from intrinsic factors, such as those determined by the structure of the molecular networks governing normal cellular physiology.</p> <p>Results</p> <p>First, simple models of single gene insufficiency using the positive feedback loops that may be derived from a three-component network were studied by computer simulation using Bionet software. The network structure is shown to affect the dynamics considerably; some networks are relatively stable even when large stochastic variations in are present, while others exhibit switch-like dynamics. In the latter cases, once the network switches over to the disease state it remains in that state permanently. Model pathways for two autosomal dominant diseases, AD polycystic kidney disease and mature onset diabetes of youth (MODY) were simulated and the results are compared to known disease characteristics.</p> <p>Conclusions</p> <p>By identifying the intrinsic mechanisms involved in the onset of AD diseases, it may be possible to better assess risk factors as well as lead to potential new drug targets. To illustrate the applicability of this study of pathway dynamics, we simulated the primary pathways involved in two autosomal dominant diseases, Polycystic Kidney Disease (PKD) and mature onset diabetes of youth (MODY). Simulations demonstrate that some of the primary disease characteristics are consistent with the positive feedback - stochastic variation theory presented here. This has implications for new drug targets to control these diseases by blocking the positive feedback loop in the relevant pathways.</p

Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral—but not the apical—plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion

Bioavailability in soils

The consumption of locally-produced vegetables by humans may be an important exposure pathway for soil contaminants in many urban settings and for agricultural land use. Hence, prediction of metal and metalloid uptake by vegetables from contaminated soils is an important part of the Human Health Risk Assessment procedure. The behaviour of metals (cadmium, chromium, cobalt, copper, mercury, molybdenum, nickel, lead and zinc) and metalloids (arsenic, boron and selenium) in contaminated soils depends to a large extent on the intrinsic charge, valence and speciation of the contaminant ion, and soil properties such as pH, redox status and contents of clay and/or organic matter. However, chemistry and behaviour of the contaminant in soil alone cannot predict soil-to-plant transfer. Root uptake, root selectivity, ion interactions, rhizosphere processes, leaf uptake from the atmosphere, and plant partitioning are important processes that ultimately govern the accumulation ofmetals and metalloids in edible vegetable tissues. Mechanistic models to accurately describe all these processes have not yet been developed, let alone validated under field conditions. Hence, to estimate risks by vegetable consumption, empirical models have been used to correlate concentrations of metals and metalloids in contaminated soils, soil physico-chemical characteristics, and concentrations of elements in vegetable tissues. These models should only be used within the bounds of their calibration, and often need to be re-calibrated or validated using local soil and environmental conditions on a regional or site-specific basis.Mike J. McLaughlin, Erik Smolders, Fien Degryse, and Rene Rietr

Revealing the missing expressed genes beyond the human reference genome by RNA-Seq

<p>Abstract</p> <p>Background</p> <p>The complete and accurate human reference genome is important for functional genomics researches. Therefore, the incomplete reference genome and individual specific sequences have significant effects on various studies.</p> <p>Results</p> <p>we used two RNA-Seq datasets from human brain tissues and 10 mixed cell lines to investigate the completeness of human reference genome. First, we demonstrated that in previously identified ~5 Mb Asian and ~5 Mb African novel sequences that are absent from the human reference genome of NCBI build 36, ~211 kb and ~201 kb of them could be transcribed, respectively. Our results suggest that many of those transcribed regions are not specific to Asian and African, but also present in Caucasian. Then, we found that the expressions of 104 RefSeq genes that are unalignable to NCBI build 37 in brain and cell lines are higher than 0.1 RPKM. 55 of them are conserved across human, chimpanzee and macaque, suggesting that there are still a significant number of functional human genes absent from the human reference genome. Moreover, we identified hundreds of novel transcript contigs that cannot be aligned to NCBI build 37, RefSeq genes and EST sequences. Some of those novel transcript contigs are also conserved among human, chimpanzee and macaque. By positioning those contigs onto the human genome, we identified several large deletions in the reference genome. Several conserved novel transcript contigs were further validated by RT-PCR.</p> <p>Conclusion</p> <p>Our findings demonstrate that a significant number of genes are still absent from the incomplete human reference genome, highlighting the importance of further refining the human reference genome and curating those missing genes. Our study also shows the importance of <it>de novo </it>transcriptome assembly. The comparative approach between reference genome and other related human genomes based on the transcriptome provides an alternative way to refine the human reference genome.</p

Biochemical mechanisms implemented by human acute myeloid leukemia cells to suppress host immune surveillance

Acute myeloid leukaemia (AML) is a blood/bone marrow cancer originating from myeloid cell precusors capable of self-renewing. AML cells implement biochemical mechanisms which allow them not only to survive, but also to successfully escape immune surveillance. ln this work, we discuss crucial molecular mechanisms used by human AML cells in order to evade immune attack