Analysis of water patterns in protein kinase binding sites

Deregulation of protein kinases is associated with numerous diseases, making them important targets for drug discovery. The majority of drugs target the catalytic site of these proteins, but due to the high level of similarity within the ATP binding sites of protein kinases, it is often difficult to achieve the required pharmacological selectivity. In this study, we describe the identification and subsequent analysis of water patterns in the ATP binding sites of 171 protein kinase structures, comprising 19 different kinases from various branches of the kinome, and demonstrate that structurally similar binding sites often have significantly different water patterns. We show that the observed variations in water patterns of different, but structurally similar kinases can be exploited in the structure-based design of potent and selective kinase inhibitors.     

A guide to defining and implementing protocols for the welfare assessment of laboratory animals: eleventh report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement

The refinement of husbandry and procedures to reduce animal suffering and improve welfare is an essential component of humane science. Successful refinement depends upon the ability to assess animal welfare effectively, and detect any signs of pain or distress as rapidly as possible, so that any suffering can be alleviated. This document provides practical guidance on setting up and operating effective protocols for the welfare assessment of animals used in research and testing. It sets out general principles for more objective observation of animals, recognizing and assessing indicators of pain or distress and tailoring these to individual projects. Systems for recording indicators, including score sheets, are reviewed and guidance is set out on determining practical monitoring regimes that are more likely to detect any signs of suffering. This guidance is intended for all staff required to assess or monitor animal welfare, including animal technologists and care staff, veterinarians and scientists. It will also be of use to members of ethics or animal care and use committees. A longer version of this document, with further background information and extra topics including training and information sharing, is available on the Laboratory Animals website.     

Definition and application of good manufacturing process-compliant production of CEA-specific chimeric antigen receptor expressing T-cells for phase I/II clinical trial

Adoptive cell therapy employing gene-modified T-cells expressing chimeric antigen receptors (CARs) has shown promising preclinical activity in a range of model systems and is now being tested in the clinical setting. The manufacture of CAR T-cells requires compliance with national and European regulations for the production of medicinal products. We established such a compliant process to produce T-cells armed with a first-generation CAR specific for carcinoembryonic antigen (CEA). CAR T-cells were successfully generated for 14 patients with advanced CEA⁺ malignancy. Of note, in the majority of patients, the defined procedure generated predominantly CD4⁺ CAR T-cells with the general T-cell population bearing an effector–memory phenotype and high in vitro effector function. Thus, improving the process to generate less-differentiated T-cells would be more desirable in the future for effective adoptive gene-modified T-cell therapy. However, these results confirm that CAR T-cells can be generated in a manner compliant with regulations governing medicinal products in the European Union.     

Predicting adult emergence of Dakota skipper and Poweshiek skipperling (Lepidoptera: Hesperiidae) in Canada

Dakota skipper (Hesperia dacotae Skinner) and Poweshiek skipperling (Oarisma poweshiek Parker) (Lepidoptera: Hesperiidae) are endemic prairie species that are threatened in Canada. Surveys during the brief adult flight period are necessary to quantify population sizes, but dates of adult emergence vary widely from year to year (up to 24 days) and populations are geographically distant from one another (150–250 km). To predict adult emergence of H. dacotae and O. poweshiek, we used local weather station data to calculate the number of degree days accumulated between March 1 and adult emergence using two different models in seven different years between 2002 and 2013. We also compared the number of degree days accumulated at the soil surface where larvae and pupae reside to those accumulated using weather station data. We recommend that surveys for Dakota skipper begin when degree day accumulations (from weather stations) reach a threshold of 575 (standard model) or 600 (double sine model) in the south central portion of Manitoba and 550 (standard model) or 575 (double sine model) in the southwest region of Manitoba. For Poweshiek skipperling surveys should be considered after degree day accumulations reach 575 (standard model) or 625 (double sine model). Degree days accumulated at the soil surface were 20–30 % greater than those calculated using weather station data in Dakota skipper sites, and 1–12 % greater in Poweshiek skipperling sites. Using our models, we predicted adult emergence to within 48 h of emergence in 2011, 2012 and 2013.     

Mesenchymally-derived Insulin-like growth factor 1 provides a paracrine stimulus for trophoblast migration

Background  

Trophoblast migration into maternal decidua is essential for normal pregnancy. It occurs in a defined time window, is spatially highly restricted, and is aberrant in some pathological pregnancies, but the control mechanisms are as yet ill-defined. At the periphery of the placenta, chorionic villi make contact with decidua to form specialised anchoring sites that feed interstitially migrating cytotrophoblast into the placental bed.     

Frequency of glucose-6-phosphate dehydrogenase (G6PD) mutations in Chinese,Filipinos, and Laotians from Hawaii

In a Hawaii Hereditary Anemia Screening Project, 4,984 participants were tested for glucose-6-phosphate dehydrogenase (G6PD) deficiency by a filter paper blood spot fluorescence test. Abnormal samples and suspected heterozygotes were checked by quantitative G6PD assay (normal 4.5 to 14 units/g Hb). G6PD was deficient (< 1.5 units/g Hb) in 188 of 2,155 males; 7 other males had low activity (1.5 to 2.8 units/g Hb). The gene frequency, estimated from males after excluding referred and related cases, was 0.037 for Chinese, 0.134 for Filipinos, and 0.203 for Laotians. Among 2,829 females tested, family data showed 111 females were obliged to be at least heterozygous, regardless of G6PD activity, and 43 others had low G6PD activity. Most heterozygotes probably remained undetected by G6PD screening. In 28 females, activity was under 10%; in another 9 females, activity was < 1.5 units/g Hb. Since only 25 homozygotes would be predicted, this apparent excess of females with deficient activity could be due to unequal X-inactivation in some heterozygotes. DNA analysis by polymerase chain reaction amplification and special analytic procedures revealed 10 different missense mutations in 75 males. The nucleotide 835 AT and 1360 CT transitions were first detected in this Hawaiian Project; we found that the nucleotide 1360 mutation was the most common cause of G6PD deficiency in Filipinos. This is the first report of G6PD screening and analysis of molecular G6PD mutations in Filipino and Laotian populations.