Using regulatory variants to detect gene-gene interactions identifies networks of genes linked to cell immortalisation.

The extent to which the impact of regulatory genetic variants may depend on other factors, such as the expression levels of upstream transcription factors, remains poorly understood. Here we report a framework in which regulatory variants are first aggregated into sets, and using these as estimates of the total cis-genetic effects on a gene we model their non-additive interactions with the expression of other genes in the genome. Using 1220 lymphoblastoid cell lines across platforms and independent datasets we identify 74 genes where the impact of their regulatory variant-set is linked to the expression levels of networks of distal genes. We show that these networks are predominantly associated with tumourigenesis pathways, through which immortalised cells are able to rapidly proliferate. We consequently present an approach to define gene interaction networks underlying important cellular pathways such as cell immortalisation.

Nonagenarian surgical admissions for the acute abdomen: who benefits?

INTRODUCTION: Patients 90 years and older form an increasing proportion of the general population. Outcomes of their acute surgical admissions are not well documented. METHODS AND MATERIALS: Surgical management of 49 consecutive nonagenarian admissions (median age: 92 years) with an acute abdomen was compared with the management and outcome of 50 younger patients (median age: 53.5) admitted with a suspected acute abdomen over the same period. RESULTS: Nonagenarian group consisted of mainly women (71% vs. 50%; p = 0.003). The use of laboratory investigations and imaging was similar for the patients aged over 90 and the younger patients, although proportionately fewer nonagenarians were investigated by abdominal CT scan (8% vs. 24%). Of the 49 nonagenarian patients admitted, only 4% (n = 2) were operated on. In contrast, 38% (n = 19) of patients aged 50-59 (p = 0.0001) underwent a surgical intervention. A much greater proportion of nonagenarians died in hospital than patients in the 50-59 comparator group (16% nonagenarians vs. 4% comparator patients; p = 0.04). The very large majority of survivors in both age groups were discharged back to their preadmission domicile [39 (95%) nonagenarians vs. 46 (96%) comparator 50-59 year group]. CONCLUSIONS: In this study, when compared with younger patients, very few nonagenarian patients (2%) with a suspected acute abdomen benefited from surgical admission. Instead, the large majority of nonagenarians either died or were discharged back to their home address without surgery.

Hotel NHS and the acute abdomen - admit first, investigate later.

AIM: To determine the financial consequences of a policy of admission first, followed by definitive investigation for patients with an admission diagnosis of suspected acute abdomen. RESULTS: Over a 1-month period, 122 patients were admitted with a suspected surgical diagnosis of acute abdomen (55 men, 67 women); age range 16-95 years (median: 56.5). Based on surgical operation required (n = 36), death after admission (n = 6, three postoperative deaths) and/or severe surgical illness (n = 17), 56 required surgical inpatient admission, while 66 did not. The patients who did not require admission spent significantly shorter time in hospital than those who required admission (median: 5 days vs. 8.5 days; p = 0.0000). Total hospital hotel and investigation cost (not including ITU or theatre costs) for all 122 patients was 330,468 pounds. Overall, 205,468 pounds was consumed by these 56 patients who required admission, while 125,000 pounds was spent on 66 patients whose clinical course did not justify admission; 92% of which was spent on hospital hotel costs and 8% on the cost of imaging and/or endoscopy. DISCUSSION AND CONCLUSION: On a national basis, emergency General Surgery admissions account for 1000 Finished Consultant Episodes per 100,000 population. The findings of this study suggest that this equates to a national NHS spend of 650 million pounds each year, for the hotel costs of patients that could arguably avoid surgical admission altogether. Continuing to admit patients with a suspected acute abdomen first and then requesting definitive investigation makes neither clinical nor economic sense.

A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.

A theoretical study of substrate-induced activation of dienelactone hydrolase.

Dienelactone hydrolase (DLH), an enzyme from the beta-ketoadipate pathway, catalyses the hydrolysis of dienelactone to maleylacetate. DLH is unusual because it is the only known naturally occurring enzyme which contains the catalytic triad Cys ... His ... Asp. This triad has previously been created artificially in the mutant serine proteases, thiol subtilisin and thiol trypsin. In both cases the mutant enzymes exhibited activities several orders of magnitude lower than the wild type enzymes; the low reactivity has generally been attributed to the inability of these enzymes to form a catalytically active thiolate anion (Cys- ... His+ ... Asp-). The crystal structure of DLH suggests that the native enzyme exists predominantly in a catalytically inert configuration; the triad cysteine is neutral and points away from the active site binding cleft. However, a crystallographic analysis of C123S DLH complexed with an isostructural inhibitor (dienelactam) indicates that substrate binding induces a prototropic rearrangement of the active site prior to catalysis which results in the formation of a highly nucleophilic thiolate anion. We have performed ab initio SCF/MP2 calculations on a relatively small portion of the active site of DLH to examine the details of this activation process. Our calculations provide supporting evidence that the conformational changes observed in the crystal structure due to inhibitor (or substrate) binding facilitate the formation of a reactive thiolate anion. In particular, substrate binding alters the position of Glu36; the carboxylate side chain of Glu36 is pushed towards C123 enabling it to abstract the thiol proton thus creating a catalytically active thiolate anion.(ABSTRACT TRUNCATED AT 250 WORDS)

A quantum mechanical study of the active site of aspartic proteinases.

We have performed ab initio self-consistent field (SCF) and configuration interaction (CI) calculations on the active site of the aspartic proteinases pepsin and endothiapepsin. The active site, which carries a formal negative charge to effect hydrolysis, was modeled as a formic acid/formate anion moiety and a water molecule, and the nearest hydrogen bonding residues (Gly34, Ser35, Gly217, and Thr218, with respect to the residue numbering in endothiapepsin) were modeled as formamide and methanol molecules. Four possible binding modes for the active-site water molecule were considered. In contrast to previous theoretical studies, we predict that the most stable form has the water molecule forming a bifurcated hydrogen bond to the inner oxygens of Asp32 and -215, with Asp32 being ionized. The calculations suggest that the water molecule prefers to bind across the shortest OD32 ... OD215 diagonal of the active-site carboxyl groups and therefore the binding mode of the water molecule for all the native aspartic proteinases can be readily predicted by measuring these distances.

Molecular modelling of DNA-antitumour drug intercalation interactions: correlation of structural and energetic features with biological properties for a series of phenylquinoline-8-carboxamide compounds.

The crystal structure of the experimental antitumour compound N-(2-dimethylaminoethyl)-2-phenylquinoline-8-carboxamide has been determined. The geometry and conformation have been used as starting points for molecular modelling of the intercalative interactions with DNA shown by the parent compound and analogues with the phenyl ring located at alternative positions on the quinoline chromophore. A molecular mechanics force field program was used for energy minimization and calculation of intermolecular (enthalphic) binding energies. The parent quinoline-8-carboxamide and derivatives with a phenyl substituent at the 4- or 5-position were judged to be poor intercalators in both structural and energetic terms. By contrast, the 2-, 3-, and 6-phenyl derivatives all had high calculated binding energies with the phenyl groups involved in stacking with DNA base pairs. The order of energies calculated for this series of compounds has been found to correlate well with both the order of experimentally derived free energies and with the in vitro cytotoxic activity.