A direct-to-biology high-throughput chemistry approach to reactive fragment screening.

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines 'direct-to-biology' high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification.

Altered ceramide metabolism is a feature in the extracellular vesicle-mediated spread of alpha-synuclein in Lewy body disorders.

Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)-collectively Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations.

Proteomics characterisation of the L929 cell supernatant and its role in BMDM differentiation.

BMDMs are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from BM are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 cell-conditioned media (LCCM) and how it affects the BMDM phenotype.

Profiling embryonic stem cell differentiation by MALDI TOF mass spectrometry: development of a reproducible and robust sample preparation workflow.

MALDI TOF mass spectrometry (MS) is widely used to characterise and biotype bacterial samples, but a complementary method for profiling of mammalian cells is still underdeveloped. Current approaches vary dramatically in their sample preparation methods and are not suitable for high-throughput studies. In this work, we present a universal workflow for mammalian cell MALDI TOF MS analysis and apply it to distinguish ground-state naïve and differentiating mouse embryonic stem cells (mESCs), which can be used as a model for drug discovery.

Mass spectrometry techniques for studying the ubiquitin system.

Post-translational control of proteins through covalent attachment of ubiquitin plays important roles in all eukaryotic cell functions. The ubiquitin system in humans consists of 2 E1, 35 E2 and >600 E3 ubiquitin ligases as well as hundreds of deubiquitylases, which reverse ubiquitin attachment. Moreover, there are hundreds of proteins with ubiquitin-binding domains that bind one of the eight possible polyubiquitin chains.

Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs).

Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays.

A Short Diastereoselective Total Synthesis of (±)-Vibralactone.

A total synthesis of the (±)-vibralactone has been achieved in 11 steps and 16% overall yield from malonic acid. Key steps include a highly diastereoselective allylation of an α-formyl ester containing an all carbon α-quaternary center, a Pd-catalyzed deallylative β-lactonization, and an aldehyde-selective Wacker oxidation of a terminal alkene.

Starting out - Transition to staff nurse can be a a test of daunting, but positive experience.

In my final weeks at university, the daunting prospect of leaving behind my comfortable nursing student status to become a qualified nurse started to dawn on me.

Synthesis and structural characterisation of the new K2NiF4-type phases, A2In0.5Sb0.5O4(A = Sr, Ba).

In this paper the synthesis and structural characterisation of two new K2NiF4-type phases, Ba2In(0.5)Sb(0.5)O4 and Sr2In(0.

The effect of bovine somatotropin and diet on somatotropin binding sites in hepatic tissue of lactating dairy cows.

In the lactating cow, galactopoiesis is stimulated by treatment with recombinant bovine somatotropin (bST) and by an improved plane of nutrition. The present study determined the interaction between these variables and examined whether a positive galactopoietic effect was accompanied by a change in hepatic binding sites for bST. Lactating dairy cows received one of three diets with increasing nutrient density; diet 1, 150 g/kg of dry matter (DM) of crude protein (CP) and 10.

In-vivo gene transfer to the uterine endometrium.

Therapeutic interventions in reproductive biology have relied largely on steroids and antisteroids which act to regulate gene expression in target tissues. Whilst their use has transformed women's lives, few conceptual advances have been made in contraceptive technology, no means identified to improve human implantation and no new strategies developed for the treatment of benign gynaecology. A novel alternative is direct gene transfer to the organ of interest.

Functional specialization in the ruminant placenta: evidence for two populations of fetal binucleate cells of different selective synthetic capacity.

Trophoblast binucleate cells (BNC) in the ruminant placenta demonstrate a characteristic development, mature structure and migratory capacity whether situated in cotyledonary or intercotyledonary regions of the placenta. However, previous immunocytochemical studies demonstrated clear differences in gene expression in granule contents of BNC according to their anatomical location with some proteins being expressed in all BNC (e.g.

Progesterone and oestrogen receptors in the decidualized mouse uterus and effects of different types of anti-progesterone treatment.

Pseudopregnant mice were treated systemically with monoclonal anti-progesterone antibody (DB3) (model 1), or progesterone receptor antagonists RU486 or ZK98,299 (ZK299) (model 2) on day 3 post coitum. On day 4, sesame oil was administered intraluminally into one uterine horn to induce decidualization. On day 7, the average mass of the oil-injected horn was 335.

Anti-progesterone effects on maternal recognition and behaviour imprinted during first pregnancy in mice.

Anti-progesterone treatment using specific anti-progesterone antibodies or a progesterone receptor (PR) antagonist during first pregnancy impairs postpartum maternal behaviour in mice. This effect is demonstrable only if the treatment is given during pregnancy but not immediately after parturition. The purpose of the present studies was to investigate if maternal behaviour is also impaired by anti-progesterone treatment in subsequent pregnancies.

Specific interactions of progestins and anti-progestins with progesterone antibodies, plasma binding proteins and the human recombinant receptor.

This structure-activity study compares the affinity of a series of progestins, progesterone metabolites and anti-progestins for a panel of monoclonal antibodies to progesterone, coypu (Myocastor coypus) or guinea pig plasma progesterone-binding proteins (PPBPs) and the human recombinant progesterone receptor A form (PR-A). The compounds tested were progesterone, Promegestone (R5020), Mifepristone (RU486), ZK98,734, Onapristone (ZK98,299), 11 alpha-hydroxyprogesterone, 11 alpha-progesterone hemisuccinate, androsterone, etiocholanolone, 5 alpha- and 5 beta-pregnane-3,20-diones, and 20 alpha- and 20 beta-hydroxyprogesterones. The Ki values for these ligands were determined by competitive binding assays using radiolabelled progesterone as the binding site ligand.

The local differential effect of prostacyclin, prostaglandin E2 and prostaglandin F2 alpha on mammary blood flow of lactating goats.

Mammary blood flow (MBF) and milk yield are closely related in dairy ruminants, but little is known about the regulation of MBF in vivo. The local effects on MBF of injections or continuous infusions into the mammary artery of prostaglandins (PG) or indomethacin (an inhibitor of prostaglandins) respectively, were investigated in surgically prepared conscious goats. Prostacyclin (PGI2) was found to be a potent stimulator of MBF which increased linearly over the dose range 50-1000 ng.

Aberrant maternal behaviour in mice treated with a progesterone receptor antagonist during pregnancy.

The rapid onset of normal maternal behaviour after parturition in mice, consisting of cleaning, warming, feeding and protection of offspring, is primed by oestrogen, progesterone and oxytocin. Previous studies showed that passive transfer of monoclonal antibodies against progesterone significantly increases the incidence of maternal rejection of pups. To test the hypothesis that aberrant maternal behaviour is due to partial progesterone withdrawal leading to hormonal imbalance during late pregnancy, maternal rejection was assessed following treatment with a progesterone receptor antagonist.

Anti-progesterone antibody administration and the impairment of postpartum maternal care in mice.

Passive transfer of a monoclonal antibody against progesterone produces a high incidence of maternal rejection in mice after recovery from antibody-induced infertility. To investigate the mechanisms involved in this reduction of maternal care, we have examined whether the effect is due to long-term exposure to antibody. Antibody was administered i.

Physiotherapy's quest for professional status in Ontario, 1950-80.

Physiotherapy is part of the new female-dominated paramedical occupations which have witnessed considerable growth in Canada since World War II. This article examines the main dimensions of physiotherapy's professionalizing drive in Ontario between 1950 and 1980. It discusses the leadership's continuing efforts to make physiotherapy a self-regulated profession, efforts which will be seriously challenged by the provincial government's growing intervention in the health sector in the 1970s.

Oestrogen synthesis by the peri-implantation conceptus of the one-humped camel (Camelus dromedarius).

The ability of the embryonic tissues of the dromedary camel (Camelus dromedarius) to synthesize oestrogens in vitro was studied in 15 conceptuses recovered by non-surgical uterine lavage between 10 and 33 days after ovulation. Definitive evidence for strong aromatase activity with synthesis of considerable quantities of oestrogens was obtained at all stages when conceptus tissues were incubated with [3H]androstenedione. A high proportion of the oestrogens was in the form of oestradiol, which contrasts to the higher ratio of oestrone:oestradiol in the oestrogens synthesized by embryonic tissues of horses and pigs.