Long-term follow-up of a multicentre cohort study on laparoscopic peritoneal lavage for perforated diverticulitis

textabstractAim Laparoscopic peritoneal lavage has increasingly been investigated as a promising alternative to sigmoidectomy for perforated diverticulitis with purulent peritonitis. Most studies only reported outcomes up to 12 months. Therefore, the objective of this study was to evaluate long-term outcomes of patients treated wit

Distance between the scissile phosphate (−19) and the centre of mass of the RNaseH active site (p66 residues 443, 478, 498 and 549) over the course of the simulations of the HIV-1 RT bound to an RNA/DNA substrate.

<p>The simulation for the wild-type A400 is shown in red, and for substitution T400 in blue. Frequency distributions of the χ₁ dihedral of p51 residue E396 in simulations of (B) RNA/DNA hybrid substrate, (C) double stranded DNA substrate and (D) NVP bound HIV-1 RT (results for the unbound RT are shown in Fig S16 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074078#pone.0074078.s001" target="_blank">File S1</a>). The simulation for A400 is shown in red, T400 in blue.</p

Association between position 400 (alanine vs threonine) and polymerase domain mutations in subtype B viruses from patients failing therapy.

*<p>p-values <0.05 are considered significant.</p

The average angle between RNaseH primer grip residues in the p51 (residues 395 and 396) and p66 (359 and 360) subunits observed in simulations of HIV-1 RT with alanine and threonine at position 400.

<p>Standard deviations are shown in parentheses.</p

Changes in the RT sequence of HIV-1 subtype B viruses that are found with increased frequency in patients failing therapy.

a<p>Correction based on the False Discovery Rate (FDR) approach.</p>b<p>p-values <0.05 are considered significant.</p

DNA polymerase activity of virus-derived RT enzymes containing substitution A400T.

<p>(A) DNA polymerase activity was measured using a 51-nt RNA template, representing the HIV-1 primer binding site (PBS) with flanking sequences. A 21-nt DNA primer complementary to the PBS was 5′-end-labeled (asterisk), and heat-annealed onto the RNA template. Extension of the primer by RT will result in a 38-nt labeled cDNA product. (B) The RNA template/DNA primer complex was incubated with the different virion-derived RTs in the presence of dNTPs. Formation of the cDNA product (cDNA) was monitored after 3, 7 and 15 min incubation. A control reaction was performed in the absence of RT (lane 19) showing only the labeled primer (P). (C) The amount of cDNA production was quantified in three independent experiments. Plotted is the average cDNA production with standard deviation (error bars). The polymerase activity of the wild-type virus-derived RT was set at 100%.</p

RNaseH activity of virus-derived RT enzymes containing substitution A400T.

<p>(A) RNaseH cleavage was measured using a 5′-end labeled (asterisk) 51-nt HIV-1 RNA template representing the PBS region. A 21-nt DNA primer complementary to the PBS was heat-annealed onto the RNA template. Primary (−19) and secondary (−12/−9) cleavages were monitored. (B) Representative gel showing RNaseH cleavage mediated by the virion-derived RTs with substitution A400T in the wild-type and M184V background. The reactions were initiated by the addition of Mg²⁺, and template (T) cleavage was monitored after 3, 7, 20 and 40 min of incubation. Control reactions were performed in the absence of Mg²⁺ (lane 18) or RT (lane 19), showing no template degradation. (C) The formation of secondary cleavage products over time was quantified, and is plotted for this representative gel. (D) RNaseH cleavage mediated by the virion-derived RTs with substitution A400T in the wild-type and TAM background was analyzed, and formation of secondary cleavage products is plotted in (E). Representative experiments are shown, and three independent experiments were performed with similar results.</p

Structure of the HIV-1 reverse transcriptase (RT) enzyme.

<p>(A) Shows RT bound to a DNA/DNA template/primer complex (shown in surface representation). The enzyme is shown in cartoon representation and coloured by subdomain; fingers are blue, palm red, thumb green, connection yellow and the RNaseH orange. The p66 subunit is shown in the foreground and in darker shades. A black rectangle indicates the location of the p51 RNaseH primer grip. (B) and (C) show detailed views of this region, including chemical representations of the residues K395 and E396 in the wild-type and A400T RT enzymes, respectively. A green line is used to illustrate the hydrogen bond formed between T400 and E396.</p

Drug susceptibility and replication of A400T viruses.

<p>(A) Susceptibility of the A400T virus to NNRTI (light grey) and NRTI (dark grey) was measured using single-cycle drug susceptibility assays. The average of three independent experiments is plotted, and the standard deviation is shown (error bars). The susceptibility of the A400T virus is shown as the relative difference in IC₅₀ compared to the wild-type pNL4.3-XN virus. (B) Replication of viruses containing A400T alone, or in combination with M184V or TAMs. SupT1 cells were infected with an equal amount of virus (50 ng). CA-p24 production was measured in the culture medium at several days post infection. A representative experiment is shown. Three independent experiments were performed with similar results.</p

A Polymorphism at Position 400 in the Connection Subdomain of HIV-1 Reverse Transcriptase Affects Sensitivity to NNRTIs and RNaseH Activity

<div><p>Reverse transcriptase (RT) plays an essential role in HIV-1 replication, and inhibition of this enzyme is a key component of HIV-treatment. However, the use of RT inhibitors can lead to the emergence of drug-resistant variants. Until recently, most clinically relevant resistance mutations were found in the polymerase domain of RT. Lately, an increasing number of resistance mutations has been identified in the connection and RNaseH domain. To further explore the role of these domains we analyzed the complete RT sequence of HIV-1 subtype B patients failing therapy. Position A/T400 in the connection subdomain is polymorphic, but the proportion of T400 increases from 41% in naïve patients to 72% in patients failing therapy. Previous studies suggested a role for threonine in conferring resistance to nucleoside RT inhibitors. Here we report that T400 also mediates resistance to non-nucleoside RT inhibitors. The susceptibility to NVP and EFV was reduced 5-fold and 2-fold, respectively, in the wild-type subtype B NL4.3 background. We show that substitution A400T reduces the RNaseH activity. The changes in enzyme activity are remarkable given the distance to both the polymerase and RNaseH active sites. Molecular dynamics simulations were performed, which provide a novel atomistic mechanism for the reduction in RNaseH activity induced by T400. Substitution A400T was found to change the conformation of the RNaseH primer grip region. Formation of an additional hydrogen bond between residue T400 and E396 may play a role in this structural change. The slower degradation of the viral RNA genome may provide more time for dissociation of the bound NNRTI from the stalled RT-template/primer complex, after which reverse transcription can resume.</p></div