Role of prophylactic midurethral sling in preventing post-operative stress urinary incontinence following repair of anterior vaginal wall prolapse

Objective: This study was conducted to find whether, among women without preoperative stress incontinence who underwent surgery for repair of anterior vaginal wall prolapse, the placement of a prophylactic midurethral mesh along with the prolapse correction surgery helped to reduce the incidence of post-operative stress urinary incontinence (POSUI). Materials & Methods: 145 women with anterior vaginal compartment prolapse were randomly assigned to receive either suitable corrective surgery for prolapse or corrective surgery along with concurrent placement of a prophylactic midurethral sling by a transobturator Prolene tape. The primary endpoint was urinary incontinence at three months and twelve months post surgery. Secondary outcomes included expected and unexpected adverse events. Results: At three months follow up the symptoms of urinary incontinence and/or positive cough test did not differ significantly between the two groups. But at twelve months, both the symptoms of urinary incontinence (9.59% versus 23.61%, p = 0.025, 95% CI = -25.93% to -2.11%, CMLE OR =0.346) and positive cough test (8.22% versus 25%, p = 0.007, 95% CI = -28.60% to -4.96%, CMLE OR = 0.271) were significantly lower in the study group compared to the control group. Expected and unexpected adverse events during operation and through the first year after surgery were comparable in both groups Conclusion: Placement of a midurethral sling by a Prolene mesh at the time of prolapse repair surgery significantly reduces the incidence of POSUI in women who were continent preoperatively. For this, the transobturator tape method is safe and effective with a low rate of complications

eIF1A/eIF5B Interaction Network and its Functions in Translation Initiation Complex Assembly and Remodeling

Eukaryotic translation initiation is a highly regulated process involving multiple steps, from 43S pre-initiation complex (PIC) assembly, to ribosomal subunit joining. Subunit joining is controlled by the G-protein eukaryotic translation initiation factor 5B (eIF5B). Another protein, eIF1A, is involved in virtually all steps, including subunit joining. The intrinsically disordered eIF1A C-terminal tail (eIF1A-CTT) binds to eIF5B Domain-4 (eIF5B-D4). The ribosomal complex undergoes conformational rearrangements at every step of translation initiation; however, the underlying molecular mechanisms are poorly understood. Here we report three novel interactions involving eIF5B and eIF1A: (i) a second binding interface between eIF5B and eIF1A; (ii) a dynamic intramolecular interaction in eIF1A between the folded domain and eIF1A-CTT; and (iii) an intramolecular interaction between eIF5B-D3 and -D4. The intramolecular interactions within eIF1A and eIF5B interfere with one or both eIF5B/eIF1A contact interfaces, but are disrupted on the ribosome at different stages of translation initiation. Therefore, our results indicate that the interactions between eIF1A and eIF5B are being continuously rearranged during translation initiation. We present a model how the dynamic eIF1A/eIF5B interaction network can promote remodeling of the translation initiation complexes, and the roles in the process played by intrinsically disordered protein segments

Translation misreading in lacZ gene in Escherichia coli

Misreading error may happen during messenger RNA translation in the ribosomal A site when there is a mismatch between the codon of mRNA and the anticodon of tRNA. Several studies have used different methods and reported varying error rates, making comparisons difficult. In this study I have developed more accurate estimates for error rates in E.coli, studying mutations affecting glutamic acid 537 (E537) and histidine 391 (H391), in the active site of the enzyme ?-galactosidase. Most mutations at the E537 (H391) codon reduce enzyme activity to 3-5 x 10-5 (4-9 x 10-5) the level of wild type. Some mutants, however, have much higher activity, and I hypothesized that this is due to misreading by tRNAGLU and . My study reports on the missense error rates of several near-cognate codons in these two residues. I also used aminoglycoside antibiotics and ribosomal protein mutations (S4, S5 and S12) to study their effects on these near and non-cognate codons and found that they affect misreading on only some near-cognate codons. These results suggest that the effect of these ribosomal protein mutations and aminoglycoside antibiotics in translation misreading are less general than has been previously reported

A single mismatch in the DNA induces enhanced aggregation of MutS: hydrodynamic analyses of the protein-DNA complexes

Changes in the oligomeric status of MutS protein was probed in solution by dynamic light scattering (DLS), and corroborated by sedimentation analyses. In the absence of any nucleotide cofactor, free MutS protein [hydrodynamic radius (Rh) of 10–12 nm] shows a small increment in size (Rh 14 nm) following the addition of homoduplex DNA (121 bp), whereas the same increases to about 18–20 nm with heteroduplex DNA containing a mismatch. MutS forms large aggregates (Rh>500 nm) with ATP, but not in the presence of a poorly hydrolysable analogue of ATP (ATPγS). Addition of either homo- or heteroduplex DNA attenuates the same, due to protein recruitment to DNA. However, the same protein/DNA complexes, at high concentration of ATP (10 mm), manifest an interesting property where the presence of a single mismatch provokes a much larger oligomerization of MutS on DNA (Rh>500 nm in the presence of MutL) as compared to the normal homoduplex (Rh≈ 100–200 nm) and such mismatch induced MutS aggregation is entirely sustained by the ongoing hydrolysis of ATP in the reaction. We speculate that the surprising property of a single mismatch, in nucleating a massive aggregation of MutS encompassing the bound DNA might play an important role in mismatch repair system

eIF1A/eIF5B interaction network and its functions in translation initiation complex assembly and remodeling

Eukaryotic translation initiation is a highly regulated process involving multiple steps, from 43S pre-initiation complex (PIC) assembly, to ribosomal subunit joining. Subunit joining is controlled by the G-protein eukaryotic translation initiation factor 5B (eIF5B). Another protein, eIF1A, is involved in virtually all steps, including subunit joining. The intrinsically disordered eIF1A C-terminal tail (eIF1A-CTT) binds to eIF5B Domain-4 (eIF5B-D4). The ribosomal complex undergoes conformational rearrangements at every step of translation initiation; however, the underlying molecular mechanisms are poorly understood. Here we report three novel interactions involving eIF5B and eIF1A: (i) a second binding interface between eIF5B and eIF1A; (ii) a dynamic intramolecular interaction in eIF1A between the folded domain and eIF1A-CTT; and (iii) an intramolecular interaction between eIF5B-D3 and -D4. The intramolecular interactions within eIF1A and eIF5B interfere with one or both eIF5B/eIF1A contact interfaces, but are disrupted on the ribosome at different stages of translation initiation. Therefore, our results indicate that the interactions between eIF1A and eIF5B are being continuously rearranged during translation initiation. We present a model how the dynamic eIF1A/eIF5B interaction network can promote remodeling of the translation initiation complexes, and the roles in the process played by intrinsically disordered protein segments

Red-backed vole brain promotes highly efficient in vitro amplification of abnormal prion protein from macaque and human brains infected with variant Creutzfeldt-Jakob disease agent.

Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrP(TSE)) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrP(TSE) in tissues and blood. Macaque vCJD PrP(TSE) did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrP(TSE). The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrP(TSE). Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrP(TSE) was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrP(TSE) demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrP(TSE) was more permissive than human PrP(TSE) in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrP(TSE) from brains of humans and macaques with vCJD. PrP(TSE) signals were reproducibly detected by Western blot in dilutions through 10⁻¹² of vCJD-infected 10% brain homogenates. This is the first report showing PrP(TSE) from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrP(TSE) in vCJD-infected human and macaque blood

PMCA with dilution series of vCJD human brain homogenate.

<p>Human vCJD brain material was diluted 10^-3-10^-12 into 10% RBVS/S saline-perfused NBH and subjected to 4 rounds of PMCA. Human vCJD 5% brain homogenate was loaded as a positive control (Cont.) in all 4 rounds. Round 1 shows the products of 10^-3-10^-7 dilutions, Round 2 shows the products of 10^-5-10^-9 dilutions, Round 3 shows the products of 10^-8-10^-12 dilutions, Round 4 shows the products of 10^-8-10^-12 dilutions. Prior to electrophoresis, samples were digested with PK. All blots were stained with 6D11 antibody except the controls lanes that were stained with 3F4 antibody.</p

Intraspecies PMCA with vCJD macaque brain homogenate.

<p>A 10% suspension of brain tissue from macaque with terminal vCJD was diluted 10^-2 or 10^-3 into 10% normal macaque brain homogenate and subjected to 1 round of PMCA (lanes 1-4). Lanes 5-8 indicate 10% macaque NBH without seed subjected to 1 round of PMCA. A 10% brain homogenate from hamsters infected with scrapie (263K) was diluted 10^-2 fold into 10% hamster NBH and subjected to 1 round of PMCA (lanes 9-10). Macaque vCJD brain homogenate (1%) was loaded in lanes 11-12 and 1% 263K scrapie hamster brain homogenate was loaded in lanes 13-14 as positive controls (Cont.). Prior to electrophoresis, samples were digested with proteinase K (PK) as indicated. The blot was stained with 3F4 antibody. Ma = macaque; Ha = hamster; PK = proteinase K.</p

PMCA with a dilution series of vCJD macaque brain homogenate.

<p>Macaque vCJD brain was diluted 10^-3-10^-12 in 10% 170 RBV 170S/S saline-perfused NBH and subjected to 4 rounds of PMCA. Macaque vCJD 5% brain homogenate was loaded as positive control (Cont.) in all 4 rounds. Round 1 shows products of the 10^-3-10^-7 dilutions, Round 2 shows the 10^-5-10^-9 dilutions, Round 3 shows the products of 10^-7-10^-11 dilutions, Round 4 shows the products of 10^-9-10^-12 dilutions. Prior to electrophoresis, samples were digested with PK. All blots were stained with 6D11 antibody except the control lanes that were stained with 3F4 antibody. </p

Interspecies PMCA with vCJD macaque brain homogenate and red-backed vole 170N/N as the substrate.

<p>Macaque vCJD brain material and hamster 263K scrapie brain material was diluted 10²-fold into 10% RBV 170N/N NBH and subjected to 1 round of PMCA (lanes 1-4 respectively). Lanes 5-6 indicate control PMCA samples containing 10% RBV 170N/N NBH substrate only without added seed. Scrapie hamster brain material was diluted 10² fold into 10% hamster NBH and subjected to 1 round of PMCA (lanes 7-8). Lanes 9-10 indicate 10% hamster NBH without seed subjected to 1 round of PMCA. Scrapie hamster brain homogenate (1%) was loaded in lanes 11^-12 as a positive control (Cont.). Prior to electrophoresis, samples were digested with PK. The blot was stained with 6D11 antibody. </p