Laparoscopic exploration of the common bile duct may be a better way of removing stones than endoscopic sphincterotomy plus laparoscopic cholecystectomy. :

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5′-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5′-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5′polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via ‘phosphate steering’, basic residues energetically steer an inverted ss 5′-flap through a gateway over FEN1’s active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5′-flap specificity and catalysis, preventing genomic instability.

SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-L-methionine to the lysine ⑀-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-L-methionine, and its non-reactive analogs Aza-adenosyl-L-methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-L-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product ⑀-N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the ⑀-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine ⑀-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation.Protein lysine methylation has emerged as a prominent post-translational modification in gene regulatory and intracellular signaling pathways. In the nucleus, site-specific methylation of lysines within histones, transcription factors, and mitotic proteins governs a diverse array of processes within the nucleus including gene expression, DNA damage checkpoint control, cell cycle progression, and mitosis (1). In 2000, a breakthrough in our understanding of this modification occurred with the discovery of the first histone-specific protein lysine methyltransferases (PKMTs) 4 (2). These enzymes possess a conserved catalytic SET domain, a 120-residue motif that was named for three Drosophila gene regulatory factors, SU(VAR)3-9, E(Z), and TRX (3). This domain shares no apparent sequence or structural homology with other S-adenosyl-L-methionine (AdoMet)-dependent enzymes, establishing the SET domain family as a novel class of methyltransferases. This seminal discovery heralded the identification of a multitude of SET domain PKMTs, which methylate histone and non-histone substrates (1).Since their identification, crystal structures of several SET domain PKMTs in complex with various substrates and products have been reported, yielding insights into the catalytic mechanism and protein substrate specificity of this methyltransferase family. These structures include the histone methyltransferases SET7/9 (4 -8), SET8 (also known as PR-SET7) (9, 10), and Neurospora DIM-5 (11) as well as pea R...

There was no difference between approaches to duct clearance in terms of postoperative stay, complications, or conversion in higher risk patients, but the laparoscopic approach was more effective and efficient and avoided unnecessary procedures.

Laparoscopic bile duct exploration is effective and safe when used for all patients. For young and fit patients it should replace endoscopic sphincterotomy.

Background:Little is known about the spontaneous passage of bile duct stones. The aim of this study was to determine the rate of spontaneous stone passage and relate it to the clinical presentation of the bile duct stone. Patients and Methods: Prospectively collected data were studied on a total of 1000 consecutive patients undergoing laparoscopic cholecystectomy with or without laparoscopic common duct exploration. Comparisons were made between 142 patients with common bile duct stones (CBDS), 468 patients who had no previous or current evidence of duct stones, and 390 patients who had good evidence of previous duct stones but none at the time of cholecystectomy. The evidence used for previous duct stones included a good history of jaundice or pancreatitis. In patients with biliary colic or cholecystitis, abnormal pre-operative liver function tests and/or a dilated common bile duct were taken as evidence of bile duct stones. Results: Of the 1000 patients studied, 532 had evidence of stones in the common bile duct at some time prior to cholecystectomy. At the time of operation, only 142 patients had bile duct stones. By implication, 80%, 84%, 93% and 55% of patients presenting with pancreatitis, colic, cholecystitis and jaundice (73% overall) had passed their bile duct stones spontaneously. All 4 patients with cholangitis had duct stones at the time of operation. Conclusions: It is likely that most bile duct stones (3 in 4) pass spontaneously, especially after pancreatitis, biliary colic and cholecystitis but less commonly after jaundice. Cholangitis appears to be always associated with the presence of duct stones at the time of operation.

Transmissible spongiform encephalopathies (TSEs) are a family of invariably fatal neurodegenerative disorders for which no effective curative therapy currently exists. We report here the synthesis of a library of indole-3-glyoxylamides and their evaluation as potential antiprion agents. A number of compounds demonstrated submicromolar activity in a cell line model of prion disease together with a defined structure-activity relationship, permitting the design of more potent compounds that effected clearance of scrapie in the low nanomolar range. Thus, the indole-3-glyoxylamides described herein constitute ideal candidates to progress to further development as potential therapeutics for the family of human prion disorders.

The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein–substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed.