DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1 Catalyzed Reaction.

Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5'-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5'-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5'-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5'-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Y40, D181 and R100 and a reacting duplex 5'-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound, 5'-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage

Pravastatin for early-onset pre-eclampsia:a randomised, blinded, placebo-controlled trial

Objective: Women with pre-eclampsia have elevated circulating levels of soluble fms-like tyrosine kinase-1 (sFlt-1). Statins can reduce sFlt-1 from cultured cells and improve pregnancy outcome in animals with a pre-eclampsia-like syndrome. We investigated the effect of pravastatin on plasma sFlt-1 levels during pre-eclampsia. Design: Blinded (clinician and participant), proof of principle, placebo-controlled trial. Setting: Fifteen UK maternity units. Population: We used a minimisation algorithm to assign 62 women with early-onset pre-eclampsia (24 +0–31 +6 weeks of gestation) to receive pravastatin 40 mg daily (n = 30) or matched placebo (n = 32), from randomisation to childbirth. Primary outcome: Difference in mean plasma sFlt-1 levels over the first 3 days following randomisation. Results: The difference in the mean maternal plasma sFlt-1 levels over the first 3 days after randomisation between the pravastatin (n = 27) and placebo (n = 29) groups was 292 pg/ml (95% CI −1175 to 592; P = 0.5), and over days 1–14 was 48 pg/ml (95% CI −1009 to 913; P = 0.9). Women who received pravastatin had a similar length of pregnancy following randomisation compared with those who received placebo (hazard ratio 0.84; 95% CI 0.50–1.40; P = 0.6). The median time from randomisation to childbirth was 9 days [interquartile range (IQR) 5–14 days] for the pravastatin group and 7 days (IQR 4–11 days) for the placebo group. There were three perinatal deaths in the placebo-treated group and no deaths or serious adverse events attributable to pravastatin. Conclusions: We found no evidence that pravastatin lowered maternal plasma sFlt-1 levels once early-onset pre-eclampsia had developed. Pravastatin appears to have no adverse perinatal effects. Tweetable abstract: Pravastatin does not improve maternal plasma sFlt-1 or placental growth factor levels following a diagnosis of early preterm pre-eclampsia #clinicaltrial finds

Shadow of a game: Locating soccer in Australian Cultural Life

This is an argument about legitimacy. Soccer—my preferred term—is at least 125 years old in Victoria, older in New South Wales. Yet it seems that proponents of the game must constantly justify themselves in watching, playing, preferring this supposedly ‘new Australian’ sport. Since 1880, soccer has sought welcome in Australian society only to be rebuffed and rejected as a foreign game, a threat, sometimes even a menace to Australian masculinity and life in general. The game has endured sustained media myopia offset by frequent outbursts of intense and spiteful attention. Johnny Warren encapsulated this anti-soccer mentality in the title of his 2002 memoir Sheilas, Wogs and Poofters. Added to this, soccer has regularly collapsed under the massive weight of war and depression and often resurged on migrant tides

Literary Publishing in a Nutshell

The article reviews several literary books including "Prochownik's Dream," by Alex Miller, "Cusp," by Josephine Wilson," An Accidental Terrorist," by Steven Lang and "Carry Me Down," by M. J. Hyland