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.     

Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells

One of the hallmarks of HIV-1 disease is the association of heightened CD4⁺ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4⁺ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4⁺ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4⁺ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4⁺ Th-cells induced by exposed DCs.     

Inactivation of NADP+-dependent isocitrate dehydrogenase by peroxynitrite. Implications for cytotoxicity and alcohol-induced liver injury

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) by supplying NADPH for antioxidant systems. We investigated whether the ICDH would be a vulnerable target of peroxynitrite anion (ONOO-) as a purified enzyme, in intact cells, and in liver mitochondria from ethanol-fed rats. Synthetic peroxynitrite and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a peroxynitrite-generating compound, inactivated ICDH in a dose- and time-dependent manner. The inactivation of ICDH by peroxynitrite or SIN-1 was reversed by dithiothreitol. Loss of enzyme activity was associated with the depletion of the thiol groups in protein. Immunoblotting analysis of peroxynitrite-modified ICDH indicates that S-nitrosylation of cysteine and nitration of tyrosine residues are the predominant modifications. Using electrospray ionization mass spectrometry (ESI-MS) with tryptic digestion of protein, we found that peroxynitrite forms S-nitrosothiol adducts on Cys305 and Cys387 of ICDH. Nitration of Tyr280 was also identified, however, this modification did not significantly affect the activity of ICDH. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by peroxynitrite. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and binding of the hydrophobic probe 8-anilino-1-napthalene sulfonic acid. When U937 cells were incubated with 100 microM SIN-1 bolus, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Using immunoprecipitation and ESI-MS, we were also able to isolate and positively identify S-nitrosylated and nitrated mitochondrial ICDH from SIN-1-treated U937 cells as well as liver from ethanol-fed rats. Inactivation of ICDH resulted in the pro-oxidant state of cells reflected by an increased level of intracellular reactive oxygen species, a decrease in the ratio of [NADPH]/[NADPH + NADP+], and a decrease in the efficiency of reduced glutathione turnover. The peroxynitrite-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Metformin modifies the exercise training effects on risk factors for cardiovascular disease in impaired glucose tolerant adults

Impaired glucose tolerant (IGT) adults are at elevated risk for cardiovascular disease (CVD). Exercise or metformin reduce CVD risk, but the efficacy of combining treatments is unclear.

Objective:

To determine the effects of exercise training plus metformin (EM), compared with each treatment alone, on CVD risk factors in IGT adults.

Design and Methods:

Subjects were assigned to placebo (P), metformin (M), exercise training plus placebo (EP), or EM (8/group). In a double‐blind design, P or 2,000 mg/d of M were administered for 12 weeks and half performed aerobic and resistance training 3 days/week for ～60 min/day at 70% pretraining heart rate peak. Outcomes included adiposity, blood pressure (BP), lipids, and high sensitivity C‐reactive protein (hs‐CRP). Z‐scores were calculated to determine metabolic syndrome severity.

Results:

M and EM, but not EP, decreased body weight compared with P (P < 0.05). M and EP lowered systolic blood pressure by 6% (P < 0.05), diastolic blood pressure by 6% (P < 0.05), and hs‐CRP by 20% (M: trend P = 0.06; EP: P < 0.05) compared with P. Treatments raised high‐density lipoprotein cholesterol (P < 0.05; EM: trend P = 0.06) compared with P and lowered triacyglycerol (P < 0.05) and metabolic syndrome Z‐score compared with baseline (EP; trend P = 0.07 and EM or M; P < 0.05).

Conclusions:

Although exercise and/or metformin improve some CVD risk factors, only training or metformin alone lowered hs‐CRP and BP. Thus, metformin may attenuate the effects of training on some CVD risk factors and metabolic syndrome severity in IGT adults.     

Classification of hybrid and altered Vibrio cholerae strains by CTX prophage and RS1 element structure

Analysis of the CTX prophage and RS1 element in hybrid and altered Vibrio cholera O1 strains showed two classifiable groups. Group I strains contain a tandem repeat of classical CTX prophage on the small chromosome. Strains in this group either contain no element(s) or an additional CTX prophage or RS1 element(s) on the large chromosome. Group II strains harbor RS1 and CTX prophage, which has an E1 Tor type rstR and classical ctxB on the large chromosome.