Manifestation of neuronal ceroid lipofuscinosis in Australian Merino sheep: observations on altered behaviour and growth.

Neuronal ceroid lipofuscinoses (NCL) is an inherited neurodegenerative disorder in children. Presently there is no effective treatment and the disorder is lethal. NCL occur in a variety of non-human species including sheep, which are recognised as valuable large animal models for NCL. This experiment investigated the progressive postural, behavioural and liveweight changes in NCL-affected lambs, to establish practical, non-invasive biomarkers of disease progression for future preclinical trials in a CLN6 Merino sheep model. A flock of eight lambs at pasture was studied, with the observer blind to the disorder status. Three genotypes were compared: homozygous affected NCL; n = 4), clinically normal heterozygous (Carrier; n = 2) and homozygous normal (non-carrier control (Normal); n = 2). Direct observation during daylight and continuous accelerometer measurements over 72 h were used to quantify lamb posture and behaviour in 11 sessions between 26-60 weeks of age, conducted at 3-5 week intervals. There was a Genotype (G) × Age (A) interaction (P = 0.001) for liveweight of the lambs in the experiment, with NCL, Carrier and Normal lambs gaining 11.8, 16.5 and 23.4 kg, respectively, between 26 and 60 weeks of age. G×A interactions were also found for walking behaviour (means for NCL, Carrier and Normal genotype groups at 26 and 60 weeks, were 1.7 and 7.9%, 3.3 and 3.1%, and 2.5 and 1.9% of observations, P = 0.008) and a composite variable of key behaviours identified in the principal components analysis (P < 0.001), with mean values for NCL lambs increasing three-fold compared to non-affected lambs as age increased. Similarly, NCL lambs became less responsive to visual and auditory stimuli as they aged. Mean responsiveness scores (out of 3) to visual stimuli for the NCL, Carrier and Normal genotypes at 26 and 60 weeks of age were 2.7 and 1.4, 2.8 and 2.9, and 3.0 and 3.0, respectively (G × A, P < 0.001). Changes in response to auditory stimuli were similar to visual stimuli. NCL lambs took more (P = 0.015) steps per 24 h than Carrier and Normal genotype lambs, but there was no G × A interaction. At 26 and 60 weeks of age respectively, NCL lambs took 2724 and 4121 steps per 24 h, compared to Carrier (1708 and 3105 steps) and Normal genotype lambs (2109 and 3506 steps). NCL lambs also performed less (P = 0.018) grazing behaviour than Carrier and Normal genotype lambs (66.5, 72.3 and 72.5% of observations for NCL, Carrier and Normal lambs, respectively). A number of behavioural changes identified in the experiment could form the basis for a protocol for monitoring and evaluation of disease progression.

SAR and evaluation of novel 5H-benzo[c][1,8]naphthyridin-6-one analogs as Aurora kinase inhibitors.

Several potent Aurora kinase inhibitors derived from 5H-benzo[c][1,8]naphthyridin-6-one scaffold were identified. A crystal structure of Aurora kinase A in complex with an initial hit revealed a binding mode of the inhibitor within the ATP binding site and provided insight for structure-guided compound optimization. Subsequent SAR campaign provided a potent and selective pan Aurora inhibitor, which demonstrated potent target modulation and antiproliferative effects in the pancreatic cell line, MIAPaCa-2. Furthermore, this compound inhibited phosphorylation of histone H3 (pHH3) in mouse bone morrow upon oral administration, which is consistent with inhibition of Aurora kinase B activity.

Identification of Clinical Candidate M2698, a Dual p70S6K and Akt Inhibitor, for Treatment of PAM Pathway-Altered Cancers.

Herein, we report the discovery of a novel class of quinazoline carboxamides as dual p70S6k/Akt inhibitors for the treatment of tumors driven by alterations to the PI3K/Akt/mTOR (PAM) pathway. Through the screening of in-house proprietary kinase library, 4-benzylamino-quinazoline-8-carboxylic acid amide 1 stood out, with sub-micromolar p70S6k biochemical activity, as the starting point for a structurally enabled p70S6K/Akt dual inhibitor program that led to the discovery of M2698, a dual p70S6k/Akt inhibitor. M2698 is kinase selective, possesses favorable physical, chemical, and DMPK profiles, is orally available and well tolerated, and displayed tumor control in multiple in vivo studies of PAM pathway-driven tumors.

Does direct referral after an abnormal smear improve patient experience?

BACKGROUND: Process-mapping of the patient pathway at the Birmingham Women's Hospital revealed that, after an abnormal smear was identified, women could experience a significant delay before referral for treatment. The direct referral policy was introduced in 2007 as part of a wider pan-Birmingham project to address this. AIM: To investigate the impact of the direct referral policy on patient experience. METHOD: An integrated survey was conducted (n=200) and collected both quantitative and qualitative data. Following the introduction of a new information booklet, the same survey was repeated (n=200). RESULTS: The findings showed that, although there was improvement in the information provided, some women stated that no information was received. DISCUSSION: This highlights the fact that, while there is a need to repeat annual patient surveys to ensure the patient pathway is as robust as possible, surveys may not capture the quality of patients' clinical experience. Suggestions are made to address this (see Implications for practice). CONCLUSION: This study shows the importance of evaluating any changes in care delivery. The direct referral policy has reduced the time it takes for women to be seen in the colposcopy department following an abnormal smear.

Pioglitazone rapidly reduces neuropathic pain through astrocyte and nongenomic PPARγ mechanisms.

Repeated administration of peroxisome proliferator-activated receptor gamma (PPARγ) agonists reduces neuropathic pain-like behavior and associated changes in glial activation in the spinal cord dorsal horn. As PPARγ is a nuclear receptor, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. However, we recently reported that a single intrathecal (i.t.) injection of pioglitazone, a PPARγ agonist, reduced hyperalgesia within 30 minutes, a time frame that is typically less than that required for genomic mechanisms. To determine the very rapid antihyperalgesic actions of PPARγ activation, we administered pioglitazone to rats with spared nerve injury and evaluated hyperalgesia. Pioglitazone inhibited hyperalgesia within 5 minutes of injection, consistent with a nongenomic mechanism. Systemic or i.t. administration of GW9662, a PPARγ antagonist, inhibited the antihyperalgesic actions of intraperitoneal or i.t. pioglitazone, suggesting a spinal PPARγ-dependent mechanism. To further address the contribution of nongenomic mechanisms, we blocked new protein synthesis in the spinal cord with anisomycin. When coadministered intrathecally, anisomycin did not change pioglitazone antihyperalgesia at an early 7.5-minute time point, further supporting a rapid nongenomic mechanism. At later time points, anisomycin reduced pioglitazone antihyperalgesia, suggesting delayed recruitment of genomic mechanisms. Pioglitazone reduction of spared nerve injury-induced increases in GFAP expression occurred more rapidly than expected, within 60 minutes. We are the first to show that activation of spinal PPARγ rapidly reduces neuropathic pain independent of canonical genomic activity. We conclude that acute pioglitazone inhibits neuropathic pain in part by reducing astrocyte activation and through both genomic and nongenomic PPARγ mechanisms.

Quantitation of indomethacin in serum and plasma using gas chromatography-mass spectrometry (GC-MS).

Indomethacin is a non-narcotic and non-steroidal anti-inflammatory drug used in the treatment of various inflammatory diseases such as rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In neonates, it is also used for induction of closure of patent ductus arteriosus (PDA). Its mechanism of action is believed to be through the inhibition of cyclooxygenase. Due to narrow therapeutic window and number of side effects, it's monitoring, particularly in neonates, is recommended. In the gas chromatography method described here, the drug is extracted from serum or plasma using methylene chloride and phosphate buffer (pH 6). The methylene chloride phase containing drug is separated and dried under stream of nitrogen. The drug is derivatized using Bis-(Trimethylsilyl)trifluoroacetamide (BSTFA) with 1% TMCS (trimethylchlorosilane). The derivatized drug is analyzed using gas chromatography-mass spectrometry. Quantitation of the drug in a sample is achieved by comparing responses of the unknown sample to the responses of the calibrators using selected ion monitoring. Meclofenamic acid is used as an internal standard.

Brequinar derivatives and species-specific drug design for dihydroorotate dehydrogenase.

Therapeutic agents brequinar sodium and leflunomide (Arava) work by binding in a hydrophobic tunnel formed by a highly variable N-terminus of family 2 dihydroorotate dehydrogenase (DHODH). The X-ray crystallographic structure of an analog of brequinar bound to human DHODH was determined. In silico screening of a library of compounds suggested another subset of brequinar analogs that do not inhibit human DHODH as potentially effective inhibitors of Plasmodium falciparum DHODH.

New tandem catalysis: preparation of cyclic enol ethers through a ruthenium-catalyzed ring-closing metathesis-olefin isomerization sequence.

Tandem reactions that proceed with a single metal catalyst precursor offer novel opportunities for developing efficient new reaction sequences. In this regard, reaction conditions have been identified that allows for a tandem ring-closing metathesis-olefin isomerization sequence catalyzed by a common ruthenium precursor. Specifically, the tandem process generates cyclic enol ethers from a variety of readily available acyclic dienes in a single reaction vessel using Grubbs' ruthenium alkylidene.