A designed photoenzyme for enantioselective [2+2] cycloadditions.

The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains1-4. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present5-12. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref. 13) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.

Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.

Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.

Synthesis and Conformational Analysis of Fluorinated Uridine Analogues Provide Insight into a Neighbouring-Group Participation Mechanism.

Fluorinated nucleoside analogues have attracted much attention as anticancer and antiviral agents and as probes for enzymatic function. However, the lack of direct synthetic methods, especially for 2',3'-dideoxy-2',3'-difluoro nucleosides, hamper their practical utility. In order to design more efficient synthetic methods, a better understanding of the conformation and mechanism of formation of these molecules is important. Herein, we report the synthesis and conformational analysis of a 2',3'-dideoxy-2',3'-difluoro and a 2'-deoxy-2'-fluoro uridine derivative and provide an insight into the reaction mechanism. We suggest that the transformation most likely diverges from the SN1 or SN2 pathway, but instead operates via a neighbouring-group participation mechanism.

Pac13 is a Small, Monomeric Dehydratase that Mediates the Formation of the 3'-Deoxy Nucleoside of Pacidamycins.

The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3'-deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine-5'-aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzyme's mechanism demonstrating that H42 is critical to the enzyme's activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3'-deoxy pacidamycin moiety with the synthetic anti-retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds.

Outcomes of 807 Thompson hip hemiarthroplasty procedures and the effect of surgical approach on dislocation rates.

The majority of displaced intracapsular fractures in our unit are managed with a Thompson hip hemiarthroplasty. Recent UK guidance from the National Institute for Health and Care Excellence has, however, advised against the continued used of the Thompson implant in patients with hip fracture. The aim of this study was to review the outcomes and complications after Thompson hip hemiarthroplasty, including the impact of modern surgical approaches and cementing, whilst controlling for confounding factors. We reviewed the outcomes following Thompson hip hemiarthroplasty from a series of 807 cases performed between April 2008 and November 2013. Of these, 721 (89.3%) were cemented and 86 (10.7%) uncemented. A total of 575 (71.3%) procedures were performed in female patients. The anterolateral approach was performed in 753 (93.3%) and the posterior approach with enhanced soft tissue repair in 54 (6.7%). Overall, there were 23 dislocations (2.9%). Dislocation following the posterior approach occurred in 13.0% (seven of 54) in comparison to 2.1% (16 of 753) with the anterolateral approach (odds ratio (OR) 8.5 (95% confidence interval (CI) 2.8-26.3), p < 0.001). Patients were discharged home in 459 cases (56.9%), to a care home or other hospital in 273 cases (33.8%). Of the total number of patients, 75 died during their admission (9.3%), and 51.8% (338 of 653) returned home within 30 days. The 30-day mortality was 7.1% (57 cases) and the 1-year mortality was 16.6% (116 of 699). We recommend against the continued use of the posterior approach in hip hemiarthroplasty, as enhanced soft tissue repair did not reduce the dislocation rates to an acceptable level in this series utilising the Thompson implant. Our findings, however, demonstrate satisfactory results for patients treated with the Thompson hip hemiarthroplasty performed through an anterolateral approach. We suggest that the continued use of this implant in a carefully selected patient cohort is justifiable.

1-Aryl-3,4-dihydroisoquinoline inhibitors of JNK3.

A series of 1-aryl-3,4-dihydroisoquinoline inhibitors of JNK3 are described. Compounds 20 and 24 are the most potent inhibitors (pIC50 7.3 and 6.9, respectively in a radiometric filter binding assay), with 10- and 1000-fold selectivity over JNK2 and JNK1, respectively, and selectivity within the wider mitogen-activated protein kinase (MAPK) family against p38alpha and ERK2. X-ray crystallography of 16 reveals a highly unusual binding mode where an H-bond acceptor interaction with the hinge region is made by a chloro substituent.

The identification of potent and selective imidazole-based inhibitors of B-Raf kinase.

A novel triarylimidazole derivative, SB-590885 (33), bearing a 2,3-dihydro-1H-inden-1-one oxime substituent has been identified as a potent and extremely selective inhibitor of B-Raf kinase.

Recent and episodic volcanic and glacial activity on Mars revealed by the High Resolution Stereo Camera.

The large-area coverage at a resolution of 10-20 metres per pixel in colour and three dimensions with the High Resolution Stereo Camera Experiment on the European Space Agency Mars Express Mission has made it possible to study the time-stratigraphic relationships of volcanic and glacial structures in unprecedented detail and give insight into the geological evolution of Mars. Here we show that calderas on five major volcanoes on Mars have undergone repeated activation and resurfacing during the last 20 per cent of martian history, with phases of activity as young as two million years, suggesting that the volcanoes are potentially still active today. Glacial deposits at the base of the Olympus Mons escarpment show evidence for repeated phases of activity as recently as about four million years ago. Morphological evidence is found that snow and ice deposition on the Olympus construct at elevations of more than 7,000 metres led to episodes of glacial activity at this height. Even now, water ice protected by an insulating layer of dust may be present at high altitudes on Olympus Mons.

The identification of clinical candidate SB-480848: a potent inhibitor of lipoprotein-associated phospholipase A2.

Modification of the pyrimidone 5-substituent in clinical candidate SB-435495 has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A(2) with sub-nanomolar potency. Cyclopentyl fused derivative 21, SB-480848, showed an enhanced in vitro and in vivo profile versus SB-435495 and has been selected for progression to man.

The discovery of SB-435495. A potent, orally active inhibitor of lipoprotein-associated phospholipase A(2) for evaluation in man.

The introduction of a functionalised amido substituent into a series of 1-(biphenylmethylacetamido)-pyrimidones has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A(2) with sub-nanomolar potency and very encouraging developability properties. Diethylaminoethyl derivative 32, SB-435495, was selected for progression to man.

Ecstasy is a dangerous drug.

Ecstasy, a dangerous psychoactive drug, has become a popular recreational drug on college campuses and dance halls in the United States, United Kingdom, and around the world. No reports on ecstasy have shown addictiveness, and some users of ecstasy claim they prefer infrequent use which is not the usual addictive pattern. Jaw clenching, bruxism, and some cardiac arrhythmias requiring medical attention have been associated with consumption of ecstasy and some fatalities. In large scale retrospective questionnaire studies of subjective experiences users claimed that they felt a gentle relaxation and openness to others and few adversive effects. In rats and monkeys ecstasy has caused depletion of the neurotransmitter serotonin in the brain but similar effects have not been identified for humans. Case reports have shown panic attacks, flashbacks, paranoia, and even fatalities. The Drug Enforcement Administration in 1985 placed ecstasy in Schedule I, the most restrictive drug category.

New studies of adults' responses to the Bender Gestalt.

The Bender-Gestalt test originated in 1936 with Lauretta Bender for evaluating perceptual and motor development of children 4 to 11 yr. old. Koppitz (1964) developed a scoring system for the test. Lacks (1984) contributed normative data for testing adults. Seven studies since Lacks' which have contributed to normative data of adults' responses to the Bender-Gestalt are reviewed here.

Capture and visualization of a catalytic RNA enzyme-product complex using crystal lattice trapping and X-ray holographic reconstruction.

We have determined the crystal structure of the enzyme-product complex of the hammerhead ribozyme by using a reinforced crystal lattice to trap the complex prior to dissociation and by employing X-ray holographic image reconstruction, a real-space electron density imaging and refinement procedure. Subsequent to catalysis, the cleavage site residue (C-17), together with its 2',3'-cyclic phosphate, adopts a conformation close to and approximately perpendicular to the Watson-Crick base-pairing faces of two highly conserved purines in the ribozyme's catalytic pocket (G-5 and A-6). We observe several interactions with functional groups on these residues that have been identified as critical for ribozyme activity by biochemical analyses but whose role has defied explanation in terms of previous structural analyses. These interactions may therefore be relevant to the hammerhead ribozyme reaction mechanism.

Cardiac disorders and antidepressant medications.

Tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors (MAOIs) have been associated with an increase in cardiovascular disorders, especially in depressed patients who have pre-existing cardiac disease. These disorders are less likely to occur when a therapeutic dosage is administered. Injuries because of falls are more likely in elderly depressed patients, and orthostatic hypotension occurs with the use of TCAs. Selective serotonin reuptake inhibitor (SSRI) antidepressants differ structurally and in side effects from TCAs and MAOIs. They appear to be effective for treatment of depression, and their side-effect profiles appear safer than those of earlier approved antidepressants used by depressed patients with cardiovascular disorders.

Psychological profile of pedophiles and child molesters.

Pedophiles and child molesters share some characteristics. Most are male, and they can be heterosexual, homosexual, or bisexual. Some prefer adult sex partners but choose children because they are available and vulnerable. The sexual abuse perpetrated may be a 1-time incident and may consist only of fondling. Penetration is unlikely with young children. Perpetrators' ages range from teens to midlife. Most victims are girls, and the perpetrator usually is a relative, friend, or neighbor. The home of the victim is often the setting for the incident. When boys are victims, sexual abuse may take place outside the home, and perpetrators may be strangers. Perpetrators of sexual abuse of children often claim that they themselves were victims of childhood sexual abuse. Psychological profiles are helpful but are compromised partly because many perpetrators are prisoners and control groups are lacking for this research.

Does a single metal ion bridge the A-9 and scissile phosphate groups in the catalytically active hammerhead ribozyme structure?

We have constructed a model structure that we believe represents the strongest possible physically and chemically reasonable representation of a hypothesized catalytically active hammerhead ribozyme structure in which a single divalent metal ion bridges the A9 and scissile phosphate groups. It has been proposed that such a structure arises from a conformational change in which the so-called ground-state structure (as observed by X-ray crystallography) rearranges in such a way that the pro-R oxygen atoms of both the A9 and scissile phosphate groups are directly coordinated by a single divalent metal ion in the transition-state of the hammerhead ribozyme cleavage reaction. We show that even the small subset of possible model structures that are consistent with these requirements, and that are stereochemically and sterically reasonable, are contradicted by experimental evidence. We also demonstrate that even a minimal subset of assumptions, i.e. that stems I and II are helical and that the two phosphate groups are coordinated by a divalent metal ion in the standard octahedral geometry, are sufficient to lead to this contradiction. We therefore conclude that such a mechanism of hammerhead ribozyme catalysis is untenable, at least in its present formulation.

Secondary structure mapping of an RNA ligand that has high affinity for the MetJ repressor protein and interference modification analysis of the protein-RNA complex.

The secondary structure of an RNA aptamer, which has a high affinity for the Escherichia coli MetJ repressor protein, has been mapped using ribonucleases and with diethyl pyrocarbonate. The RNA ligand is composed of a stem-loop with a highly structured internal loop. Interference modification showed that the bases within the internal loop, and those directly adjacent to it, are important in the binding of the RNA ligand to MetJ. Most of the terminal stem-loop could be removed with little effect on the binding. Ethylation interference suggests that none of the phosphate groups are absolutely essential for tight binding. The data suggest that the MetJ binding site on the aptamer is distinct from that of the natural DNA target, the 8-base pair Met box.