DNA end recognition by the Mre11 nuclease dimer: insights into resection and repair of damaged DNA

The Mre11–Rad50–Nbs1 (MRN) complex plays important roles in sensing DNA damage, as well as in resecting and tethering DNA ends, and thus participates in double-strand break repair. An earlier structure of Mre11 bound to a short duplex DNA molecule suggested that each Mre11 in a dimer recognizes one DNA duplex to bridge two DNA ends at a short distance. Here, we provide an alternative DNA recognition model based on the structures of Methanococcus jannaschii Mre11 (MjMre11) bound to longer DNA molecules, which may more accurately reflect a broken chromosome. An extended stretch of B-form DNA asymmetrically runs across the whole dimer, with each end of this DNA molecule being recognized by an individual Mre11 monomer. DNA binding induces rigid-body rotation of the Mre11 dimer, which could facilitate melting of the DNA end and its juxtaposition to an active site of Mre11. The identified Mre11 interface binding DNA duplex ends is structurally conserved and shown to functionally contribute to efficient resection, non-homologous end joining, and tolerance to DNA-damaging agents when other resection enzymes are absent. Together, the structural, biochemical, and genetic findings presented here offer new insights into how Mre11 recognizes damaged DNA and facilitates DNA repair.     

The Long-Term Influence of Body Mass Index on the Success Rate of Mid-Urethral Sling Surgery among Women with Stress Urinary Incontinence or Stress-Predominant Mixed Incontinence: Comparisons between Retropubic and Transobturator Approaches

Objectives

Mid-urethral sling (MUS) surgery for the treatment of urinary incontinence has been widespread since the introduction of tension-free vaginal tape in the mid-1990s. The majority of studies with short-term follow-up <2 years found no differences in the surgical outcomes according to body mass index (BMI). However, considering the chronic influence of obesity on pelvic floor musculature, it is cautiously speculated that higher BMI could increase stress on pelvic floor and sub-urethral tape, possibly decreasing the long-term success rate in the obese population. We aimed to compare the long-term effects of BMI on the outcomes of MUS between women with retropubic and transobturator approaches.

Methods

We performed a retrospective analysis on 243 consecutive women who received MUS and were followed up for ≥36 months. The influence of BMI on the success rates was separately estimated and the factors for treatment failure were examined using logistic regression in either approach.

Results

The mean follow-up was 58.4 months, and 30.5% were normal weight, 51.0% overweight, and 18.5% obese. Patients received either the retropubic (30.5%) or transobturator (69.5%) approach. The success rates (%) under the transobturator approach differed according to the BMI groups (94.3, 88.6, and 78.6, respectively; P = 0.037) while those under the retropubic approach were not different according to the BMI groups. However, in multivariate models, only the presence of preoperative mixed urinary incontinence (MUI) was proven to be the risk factor for treatment failure in the transobturator approach (OR 6.39, P = 0.003). The percent of subjects with MUI was higher in obese women than in non-obese women with the transobturator approach.

Conclusions

BMI was not independently associated with failures in either approach. Higher success rates in women with lower BMI in the transobturator approach were attributed to the lower percent of preoperative MUI in those with lower BMI.     

Tonsil-derived mesenchymal stem cells alleviate concanavalin A-induced acute liver injury

Acute liver failure, the fatal deterioration of liver function, is the most common indication for emergency liver transplantation, and drug-induced liver injury and viral hepatitis are frequent in young adults. Stem cell therapy has come into the limelight as a potential therapeutic approach for various diseases, including liver failure and cirrhosis. In this study, we investigated therapeutic effects of tonsil-derived mesenchymal stem cells (T-MSCs) in concanavalin A (ConA)- and acetaminophen-induced acute liver injury. ConA-induced hepatitis resembles viral and immune-mediated hepatic injury, and acetaminophen overdose is the most frequent cause of acute liver failure in the United States and Europe. Intravenous administration of T-MSCs significantly reduced ConA-induced hepatic toxicity, but not acetaminophen-induced liver injury, affirming the immunoregulatory capacity of T-MSCs. T-MSCs were successfully recruited to damaged liver and suppressed inflammatory cytokine secretion. T-MSCs expressed high levels of galectin-1 and -3, and galectin-1 knockdown which partially diminished interleukin-2 and tumor necrosis factor α secretion from cultured T-cells. Galectin-1 knockdown in T-MSCs also reversed the protective effect of T-MSCs on ConA-induced hepatitis. These results suggest that galectin-1 plays an important role in immunoregulation of T-MSCs, which contributes to their protective effect in immune-mediated hepatitis. Further, suppression of T-cell activation by frozen and thawed T-MSCs implies great potential of T-MSC banking for clinical utilization in immune-mediated disease.     

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak^−/− MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak^−/− MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak^−/− MEF cells (Ahnak^−/−-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak^−/−-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak^−/− MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.     

Evaluation of pilot-scale modified A2O processes for the removal of nitrogen compounds from sewage

In this study, pilot-scale experiments for the removal of nitrogen from sewage obtained from a county Y sewer system were performed using modified A₂O processes. Using this approach, the total amount of nitrogen discharged during denitrification of the influent was average 38.6 mg/L and a level of average 10.8 mg/L was maintained throughout the denitrification process, which resulted in an average removal efficiency that was greater than 72%. The nitrogen components in the effluent water consisted of 22% ammonia nitrogen, 6% nitrite nitrogen and 72% nitrate nitrogen, reaching a nitrification efficiency of 94%. In conclusion, since these advanced treatment methods, which involve modified A₂O processes, were successfully employed to remove nitrogen from sewage discharge, they hold promise for wide spread use by treatment plants.     

Fabrication of hyaluronic acid hydrogel beads for cell encapsulation

Hyaluronic acid (HA) hydrogel beads were prepared by photopolymerization of methacrylated HA and N-vinylpyrrolidone using alginate as a temporal spherical mold. Various fabrication conditions for preparing the hydrogel beads, such as the concentration of methacrylated HA and UV irradiation time, were optimized to control swelling properties and enzymatic degradability. A new concept for cell encapsulation is proposed in this paper. Viable cells were directly injected into the hydrogel beads using a microinjection technique. When bovine articular chondrocytes were injected into HA hydrogel beads and cultivated for 1 week, the cells could proliferate well within the HA beads. HA hydrogel beads could be potentially used as injectable cell delivery vehicles for regenerating tissue defects.     

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.     

Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

The 2a (polymerase) protein of cucumber mosaic virus (CMV) was shown to be phosphorylated both in vivo and in vitro. In vitro assays using 2a protein mutants and tobacco protein kinases showed that the 2a protein has at least three phosphorylation sites, one of which is located within the N-terminal 126 amino acid region. This region is essential and sufficient for interaction with the CMV 1a protein. When phosphorylated in vitro, the 2a protein N-terminal region failed to interact with the 1a protein. Since the 1a-2a interaction is essential for the replication of CMV, this suggests that phosphorylation of the N-terminal region of the 2a protein negatively modulates the interaction in vivo, and may have a regulatory role acting directly in viral infection.