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 Ocular Benefits of Estrogen Replacement Therapy: A Population-Based Study in Postmenopausal Korean Women

Purpose

To elucidate the prevalence of cataract, glaucoma, pterygia, and diabetic retinopathy among Korean postmenopausal women with or without estrogen replacement therapy (ERT).

Methods

A cross-sectional, nationally representative sample from the 4th Korea National Health and Nutrition Examination Survey (KNHANES IV) (2007–2009) was used. Participants were interviewed for the determination of socioeconomic and gynecologic factors. Each woman also underwent an ophthalmologic examination and provided a blood sample for risk factor assessment.

Results

Of 3968 postmenopausal women enrolled, 3390 had never received estrogen, and 578 were undergoing estrogen treatment. After adjusting for age, diabetes, hypertension, high cholesterol levels, and high low-density lipoprotein levels, the prevalence of anterior polar cataract, retinal nerve fiber layer (RNFL) defect, and flesh pterygium was higher in the non-ERT group (OR, 3.24; 95% CI, 1.12–9.35, OR 1.70; 95% CI, 1.04–2.78, OR 3.725; 95% CI, 1.21–11.45, respectively). Further, the prevalence of atrophic pterygium was lower in the non-ERT group compared to that in the ERT group (OR, 0.21, 95% CI, 0.07–0.63).

Conclusions

These data suggest that ERT has a protective effect against the development of anterior polar cataract, flesh pterygium, and RNFL defect.     

Improving the accuracy of genomic prediction in Chinese Holstein cattle by using one-step blending

Background

The one-step blending approach has been suggested for genomic prediction in dairy cattle. The core of this approach is to incorporate pedigree and phenotypic information of non-genotyped animals. The objective of this study was to investigate the improvement of the accuracy of genomic prediction using the one-step blending method in Chinese Holstein cattle.

Findings

Three methods, GBLUP (genomic best linear unbiased prediction), original one-step blending with a genomic relationship matrix, and adjusted one-step blending with an adjusted genomic relationship matrix, were compared with respect to the accuracy of genomic prediction for five milk production traits in Chinese Holstein. For the two one-step blending methods, de-regressed proofs of 17 509 non-genotyped cows, including 424 dams and 17 085 half-sisters of the validation cows, were incorporated in the prediction model. The results showed that, averaged over the five milk production traits, the one-step blending increased the accuracy of genomic prediction by about 0.12 compared to GBLUP. No further improvement in accuracies was obtained from the adjusted one-step blending over the original one-step blending in our situation. Improvements in accuracies obtained with both one-step blending methods were almost completely contributed by the non-genotyped dams.

Conclusions

Compared with GBLUP, the one-step blending approach can significantly improve the accuracy of genomic prediction for milk production traits in Chinese Holstein cattle. Thus, the one-step blending is a promising approach for practical genomic selection in Chinese Holstein cattle, where the reference population mainly consists of cows.     

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.     

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.     

Activation of brain steroidogenesis and neurogenesis during the gonadal differentiation in protandrous black porgy,Acanthopagrus schlegelii

The early brain development, at the time of gonadal differentiation was investigated using a protandrous teleost, black porgy. This natural model of monosex juvenile fish avoids the potential complexity of sexual dimorphism. Brain neurogenesis was evaluated by histological analyses of the diencephalon, at the time of testicular differentiation (in fish between 90 and 150 days after hatching). Increases in the number of both Nissl‐stained total brain cells, and Pcna‐immunostained proliferative brain cells were observed in specific area of the diencephalon, such as ventromedialis thalami and posterior preoptic area, revealing brain cell proliferation. qPCR analyses showed significantly higher expression of the radial glial cell marker blbp and neuron marker bdnf. Strong immunohistochemical staining of Blbp and extended cellular projections were observed. A peak expression of aromatase (cyp19a1b), as well as an increase in estradiol (E₂) content were also detected in the early brain. These data demonstrate that during gonadal differentiation, the early brain exhibits increased E₂ synthesis, cell proliferation, and neurogenesis. To investigate the role of E₂ in early brain, undifferentiated fish were treated with E₂ or aromatase inhibitor (AI). E₂ treatment upregulated brain cyp19a1b and blbp expression, and enhanced brain cell proliferation. Conversely, AI reduced brain cell proliferation. Castration experiment did not influence the brain gene expression patterns and the brain cell number. Our data clearly support E₂ biosynthesis in the early brain, and that brain E₂ induces neurogenesis. These peak activity patterns in the early brain occur at the time of gonad differentiation but are independent of the gonads. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 121–136, 2016     

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.     

Inter- and intra-genetic variation of four wild populations of Prosopis using rapd-pcr fingerprints

The species of Prosopis (Leguminosae) are welladapted to grow in the arid regions of the world. In Mexico, nearly 70%of its territory accounts for arid lands and nine species of this genus arefound in the country which are currently utilized for multiple purposes. Giventhat a sustainable exploitation of this important natural resource is necessary,knowledge of the genetic variability of natural populations is required. Thegenetic variation among and within four wild populations of Prosopisglandulosa, P. juliflora, and two ofP. laevigata, located in the Mexican States of Chihuahua(1), Sinaloa (2), Guanajuato (3) and Hidalgo (4), respectively, was determinedusing random amplified polymorphic DNA (RAPDs). There was a genetic variation of72.48% among these four populations, and within them the estimated geneticvariation was 27.52%. Between the populations of P.laevigata the genetic variation obtained was 7.15%, and within them92.85% (AMOVA, P < 0.001). Based on the assumption ofWright's island model, the genetic variation found among populations correspondsto a gene flow (N_em) of 0.09, while the gene flow detected betweenthe populations of P. laevigata was 3.2. A total of 27 RAPDphenotypes were identified in the four populations; among them, 11 correspondedto P. laevigata. The phylogenetic analyses showed that thethree species are differentiated in three clades and that probably P.juliflora was the origin of the other two species. The protogynoustrait of their flowers and the outcrossing are the main factors that may explainthe genetic variation observed in these populations.     

Neuronal Control of Exocytosis and Calcium Homeostasis

We showed that 5 M acetylcholine (ACh) and 100 M norepinephrine (NE) cause increases in the total Ca²⁺ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 M ACh and 100 M NE increased the free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca²⁺-free external solution caused a [Ca²⁺] _i increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca²⁺ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated.