Functional Characterization of a Novel Family of Acetylcholine-Gated Chloride Channels in Schistosoma mansoni

Acetylcholine is the canonical excitatory neurotransmitter of the mammalian neuromuscular system. However, in the trematode parasite Schistosoma mansoni, cholinergic stimulation leads to muscle relaxation and a flaccid paralysis, suggesting an inhibitory mode of action. Information about the pharmacological mechanism of this inhibition is lacking. Here, we used a combination of techniques to assess the role of cholinergic receptors in schistosome motor function. The neuromuscular effects of acetylcholine are typically mediated by gated cation channels of the nicotinic receptor (nAChR) family. Bioinformatics analyses identified numerous nAChR subunits in the S. mansoni genome but, interestingly, nearly half of these subunits carried a motif normally associated with chloride-selectivity. These putative schistosome acetylcholine-gated chloride channels (SmACCs) are evolutionarily divergent from those of nematodes and form a unique clade within the larger family of nAChRs. Pharmacological and RNA interference (RNAi) behavioral screens were used to assess the role of the SmACCs in larval motor function. Treatment with antagonists produced the same effect as RNAi suppression of SmACCs; both led to a hypermotile phenotype consistent with abrogation of an inhibitory neuromuscular mediator. Antibodies were then generated against two of the SmACCs for use in immunolocalization studies. SmACC-1 and SmACC-2 localize to regions of the peripheral nervous system that innervate the body wall muscles, yet neither appears to be expressed directly on the musculature. One gene, SmACC-1, was expressed in HEK-293 cells and characterized using an iodide flux assay. The results indicate that SmACC-1 formed a functional homomeric chloride channel and was activated selectively by a panel of cholinergic agonists. The results described in this study identify a novel clade of nicotinic chloride channels that act as inhibitory modulators of schistosome neuromuscular function. Additionally, the iodide flux assay used to characterize SmACC-1 represents a new high-throughput tool for drug screening against these unique parasite ion channels.     

The design and synthesis of indazole and pyrazolopyridine based glucokinase activators for the treatment of Type 2 diabetes mellitus

Glucokinase activators represent a promising potential treatment for patients with Type 2 diabetes. Herein, we report the identification and optimization of a series of novel indazole and pyrazolopyridine based activators leading to the identification of 4-(6-(azetidine-1-carbonyl)-5-fluoropyridin-3-yloxy)-2-ethyl-N-(5-methylpyrazin-2-yl)-2H-indazole-6-carboxamide (42) as a potent activator with favorable preclinical pharmacokinetic properties and in vivo efficacy.     

Extent of invasion of Tasmanian native vegetation by the exotic bumblebee Bombus terrestris (Apoidea: Apidae)

Abstract Observations of the large earth bumblebee, Bombus terrestris (L.), in native vegetation were collated to determine the extent to which this exotic species has invaded Tasmanian native vegetation during the first 9 years after its introduction. The range of B. terrestris now encompasses all of Tasmania's major vegetation types, altitudes from sea level to 1260m a.s.L, and the entire breadth of annual precipitation in the state from more than 3200 mm to less than 600 mm. Observations of workers carrying pollen, together with the presence of large numbers of bumblebees at many localities across this range indicate that colonies are frequently established in native vegetation. Evidence that colonies are often successful was obtained from repeated observations of the species during more than 1 year at particular sites. Unequivocal evidence of colonies was obtained from six National Parks, including four of the five in the Tasmanian Wilderness World Heritage Area (WHA). Indeed, the species has been present in the WHA for at least as long as it has in the city of Hobart, where it was first recorded. In southwestern Tasmania, evidence of colonies was obtained up to 40km from gardens, 61 km from small towns and 93 km from large towns. Hence, contrary to previous suggestions, the species is established in the most remote parts of Tasmania and is not dependent on introduced garden plants. Given their strong record of invasion, it is likely that B. terrestris will form feral populations on the mainland of Australia and in many other parts of the world if introduced. Because of their likely negative impacts on native animals and plants, and potential to enhance seed production in weeds, the spread of bumblebees should be avoided.     

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

NMR and temperature-jump measurements of de novo designed proteins demonstrate rapid folding in the absence of explicit selection for kinetics

We address the importance of natural selection in the origin and maintenance of rapid protein folding by experimentally characterizing the folding kinetics of two de novo designed proteins, NC3-NCAP and ENH-FSM1. These 51 residue proteins, which adopt the helix-turn-helix homeodomain fold, share as few as 12 residues in common with their most closely related natural analog. Despite the replacement of up to 3/4 of their residues by a computer algorithm optimizing only thermodynamic properties, the designed proteins fold as fast or faster than the 35,000 s(-1) observed for the closest natural analog. Thus these de novo designed proteins, which were produced in the complete absence of selective pressures or design constraints explicitly aimed at ensuring rapid folding, are among the most rapidly folding proteins reported to date.     

Chemoirradiation for Glioblastoma Multiforme: The National Cancer Institute Experience

Purpose

Standard treatment for glioblastoma (GBM) is surgery followed by radiation (RT) and temozolomide (TMZ). While there is variability in survival based on several established prognostic factors, the prognostic utility of other factors such as tumor size and location are not well established.

Experimental Design

The charts of ninety two patients with GBM treated with RT at the National Cancer Institute (NCI) between 1998 and 2012 were retrospectively reviewed. Most patients received RT with concurrent and adjuvant TMZ. Topographic locations were classified using preoperative imaging. Gross tumor volumes were contoured using treatment planning systems utilizing both pre-operative and post-operative MR imaging.

Results

At a median follow-up of 18.7 months, the median overall survival (OS) and progression-free survival (PFS) for all patients was 17.9 and 7.6 months. Patients with the smallest tumors had a median OS of 52.3 months compared to 16.3 months among patients with the largest tumors, P = 0.006. The patients who received bevacizumab after recurrence had a median OS of 23.3 months, compared to 16.3 months in patients who did not receive it, P = 0.0284. The median PFS and OS in patients with periventricular tumors was 5.7 and 17.5 months, versus 8.9 and 23.3 months in patients with non-periventricular tumors, P = 0.005.

Conclusions

Survival in our cohort was comparable to the outcome of the defining EORTC-NCIC trial establishing the use of RT+TMZ. This study also identifies several potential prognostic factors that may be useful in stratifying patients.     

Identification and SAR of squarate inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK-2)

A novel series of inhibitors for mitogen activated protein kinase-activated protein kinase 2 (MK-2) are reported. These squarate based inhibitors were identified via a high-throughput screen. An MK2 co-structure with the starting ligand was obtained and a structure based approach was followed to optimize potency and selectivity.     

Steroid control of gonadotropin-releasing hormone secretion: associated changes in pro-opiomelanocortin and preproenkephalin messenger RNA expression in the ovine hypothalamus

The endogenous opioid peptides have been implicated in mediating the actions of estrogen and progesterone on GnRH release. We used in situ hybridization histochemistry to determine whether steroid-induced changes in GnRH/LH release in the female sheep are associated with changes in the cellular mRNA content of the precursors for beta-endorphin (pro-opiomelanocortin; POMC) and met-enkephalin (pre-proenkephalin; PENK). Two specific hypotheses were tested. First, that the inhibitory actions of progesterone are associated with an increase in opioid gene expression in specific hypothalamic nuclei. Our data support this hypothesis. Thus, an increase in progesterone was associated with increased POMC gene expression in the arcuate nucleus and PENK in the paraventricular nucleus. Further, the increase in POMC was restricted to regions of the arcuate nucleus that contain steroid sensitive beta-endorphin neurons. Our second hypothesis, that gene expression for the two opioid precursors would decrease prior to the start of the estradiol-stimulated GnRH surge, was not supported. Rather, POMC (but not PENK) gene expression in the arcuate nucleus was significantly higher in estradiol-treated animals than controls at the peak of the GnRH surge. These data suggest that beta-endorphin neurons in subdivisions of the arcuate nucleus and enkephalin neurons in the paraventricular nucleus are part of the neural network by which progesterone inhibits LH release. While enkephalin neurons may not play a role in estrogen positive feedback, increases in POMC mRNA in the arcuate nucleus at the time of the GnRH peak may be important for replenishing beta-endorphin stores and terminating estrous behavior.