Optimisation of the Schizosaccharomyces pombe urg1 expression system

The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down P urg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining P urg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair

Chronic y-secretase inhibition reduces amyloid plaque-associated instability of pre- and postsynaptic structures

The loss of synapses is a strong histological correlate of the cognitive decline in Alzheimer’s disease (AD). Amyloid bpeptide (Ab), a cleavage product of the amyloid precursor protein (APP), exerts detrimental effects on synapses, a process thought to be causally related to the cognitive deficits in AD. Here, we used in vivo two-photon microscopy to characterize the dynamics of axonal boutons and dendritic spines in APP/Presenilin 1 (APPswe/PS1L166P)–green fluorescent protein (GFP) transgenic mice. Time-lapse imaging over 4 weeks revealed a pronounced, concerted instability of pre- and postsynaptic structures within the vicinity of amyloid plaques. Treatment with a novel sulfonamide-type g-secretase inhibitor (GSI) attenuated the formation and growth of new plaques and, most importantly, led to a normalization of the enhanced dynamics of synaptic structures close to plaques. GSI treatment did neither affect spines and boutons distant from plaques in amyloid precursor protein/presenilin 1-GFP (APPPS1-GFP) nor those in GFP-control mice, suggesting no obvious neuropathological side effects of the drug

CsF–Al<SUB>2</SUB>O<SUB>3</SUB> mediated rapid condensation of phenols with aryl halides: comparative study of conventional heating vs. microwave irradiation

Biaryl ethers and thio ethers are formed in high yields by the condensation of phenols and thiophenols with electron-deficient aryl halides using CsF supported on Al2O3 under microwave irradiation in solvent-free conditions

A novel gold nanoparticle-based approach for the rapid diagnosis of meningococcal infection

The bacterial meningitis caused by Neisseria meningitidis is responsible for considerable morbidity and mortality throughout the world. Given the limitations of existing diagnostic tests and the severity of the illness associated with the disease, there is a clear requirement for a rapid and specific diagnostic assay. This thesis describes the development of nanoparticle based tests for the detection of Neisseria meningitidis specific cell surface markers. As an initial target antigen, a recombinant form of highly conserved outer membrane protein, OMP85 was used. Within the OMP85 protein sequence, a predicted antigenic sequence between residues 720 and 745 was identified and found to be unique to this organism. This amino acid sequence was synthesised as peptide (SR1) with a gly-gly-cysteine spacer sequence at the N-terminus using t-boc chemistry. Also, the major virulence factor, capsular polysaccharide of N. meningitidis serogroup B bacteria was purified. Polyclonal antibodies were raised against purified OMP85 antigen in rabbits and against SR1 peptide and also against formalin inactivated N. meningitidis serogroup B whole cell bacteria in sheep. This panel of different antibodies including the commercial anti-capsular monoclonal antibodies were examined for cross reactivity against a range of closely related Gram negative bacteria. Based on these cross-reactivity studies, a highly specific anti-NM antibody was developed following purification of the anti-SR1 antiserum by immuno-affinity chromatography. Purified OMP85 antigen and anti-OMP85 antibody were successfully conjugated on 13, 30, 40, 50 and 60 nm gold nanoparticles by an electrostatic adsorption method. Coupling of the gold nanoparticles results in a shift of the respective surface plasmon peak toward longer wavelengths (in the range of 600-800 nm) resulting in a change of the colour of the colloidal suspension from red to purple to blue. An attempt was made to develop a rapid diagnostic assay based on gold nanoparticle induced colour shift assay for N. meningitidis by utilising the specific interaction of OMP85 and anti-OMP85 antibody conjugated to gold nanoparticles as a model system. However, this system was not reproducible and is likely to be due to problems with stability of gold nanoparticles during the conjugation process. As an alternative approach, a highly selective quartz crystal microbalance (QCM)-based immunosensor was designed using the same OMP85/anti-OMP85 antibody system. A method was developed using polyvinylidene fluoride (PVDF) coated QCM crystals with protein A for the directional orientation of the antibodies. To further enhance the sensitivity of the test, OMP85-conjugated gold nanoparticles were used as signal amplification probes for the reproducible detection of the target down to 300 ng/mL, corresponding to a five fold increase in sensitivity compared to detection of OMP85 antigen alone. Also, this sensor has successfully been employed to detect whole cell bacteria at a concentration as low as 100 cfu/mL. Thus, in this study using the real-time QCM measurements, a novel strategy has been developed for the sensitive detection of both N. meningitidis bacteria and the protein antigen at very low concentrations, using gold nanoparticles as signal amplification probes

University Choir Treble Choir

Kemp Recital Hall Friday Evening April 16, 1993 8:00p

Children's thoughts and feelings about their donor and security of attachment to their solo mothers in middle childhood

$\textbf{STUDY QUESTION}$ What is the relationship between children's thoughts and feelings about their donor and their security of attachment to their solo mothers in middle childhood? $\textbf{SUMMARY ANSWER}$ Children with higher levels of secure–autonomous attachment to their mothers were more likely to have positive perceptions of the donor, and those with higher levels of insecure–disorganized attachment to their mothers were more likely to perceive him negatively. $\textbf{WHAT IS KNOWN ALREADY}$ There is limited understanding of the factors that contribute to children's thoughts and feelings about their donor in solo mother families. In adolescence, an association was found between adolescents’ curiosity about donor conception and their security of attachment to their mothers. $\textbf{STUDY DESIGN, SIZE, DURATION}$ 19 children were administered the Friends and Family Interview and Donor Conception Interview between December 2015 and March 2016 as part of the second phase of a longitudinal, multi-method, multi-informant study of solo mother families. $\textbf{PARTICIPANTS/MATERIALS, SETTING, METHODS}$ All children were aged between 7 and 13 years and had been conceived by donor insemination to solo mothers. Interviews were conducted in participants’ homes. The Friends and Family Interview was rated according to a standardized coding scheme designed to measure security of attachment in terms of secure–autonomous, insecure–dismissing, insecure–preoccupied and insecure–disorganized attachment patterns. Quantitative analyses of the Donor Conception Interview yielded two factors: interest in the donor and perceptions of the donor. Qualitative analyses of the Donor Conception Interview were conducted using qualitative content analysis and thematic analysis. $\textbf{MAIN RESULTS AND THE ROLE OF CHANCE}$ Statistically significant associations were found between the perception of the donor scale and the secure–autonomous and insecure–disorganized attachment ratings. Children with higher levels of secure–autonomous attachment to their mothers were more likely to have positive perceptions of the donor (r = 0.549, P = 0.015), and those with higher levels of insecure–disorganized attachment to their mothers were more likely to perceive him negatively (r = −0.632, P = 0.004). Children's narratives about the donor depicted him as a stranger (n = 8), a biological father (n = 4), a social parent (n = 3), or in ambivalent terms (n = 4). $\textbf{LIMITATIONS, REASONS FOR CAUTION}$ Findings are limited by the wide age range of children within a small overall sample size. Participants were those willing and able to take part in research on donor conception families. The statistical significance of correlation coefficients was not corrected for multiple comparisons. $\textbf{WIDER IMPLICATIONS OF THE FINDINGS}$ Findings highlight the importance of situating children's ideas about the donor within family contexts. It is recommended that those working with donor conception families consider this when advising parents about whether, what and how to tell children about donor conception.This study was funded by the Wellcome Trust [097857/Z/11/Z]

Metal triflates catalyzed efficient synthesis of 3,4-dihydro-2H-1-benzopyrans

Ytterbium triflate efficiently catalyzes an unusual cyclization of o-hydroxybenzaldehydes with 2,3-dihydrofuran and 3,4-dihydro-2H-pyran in the presence of trimethyl orthoformate at ambient temperature to afford a new class of compounds, furo- and pyrano[2,3-b]benzopyrans in excellent yields with high diastereoselectivity. Also, o-hydroxybenzaldehydes reacted smoothly with acetophenones in the presence of a catalytic amount of scandium triflate under similar reaction conditions to give the corresponding 2,4-dialkoxy-2-aryl-3,4-dihydro-2H-1-benzopyrans in high yields