MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals

MDC1 functions in checkpoint activation and DNA repair following DNA damage. To address the physiological role of MDC1, we disrupted the MDC1 gene in mice. MDC1-/- mice recapitulated many phenotypes of H2AX-/- mice, including growth retardation, male infertility, immune defects, chromosome instability, DNA repair defects, and radiation sensitivity. At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. In the absence of MDC1, many downstream ATM signaling events are defective. These results suggest that MDC1, as a signal amplifier of the ATM pathway, is vital in controlling proper DNA damage response and maintaining genomic stability.     

H2AX prevents DNA breaks from progressing to chromosome breaks and translocations

Histone H2AX promotes DNA double-strand break (DSB) repair and immunoglobulin heavy chain (IgH) class switch recombination (CSR) in B-lymphocytes. CSR requires activation-induced cytidine deaminase (AID) and involves joining of DSB intermediates by end joining. We find that AID-dependent IgH locus chromosome breaks occur at high frequency in primary H2AX-deficient B cells activated for CSR and that a substantial proportion of these breaks participate in chromosomal translocations. Moreover, activated B cells deficient for ATM, 53BP1, or MDC1, which interact with H2AX during the DSB response, show similarly increased IgH locus breaks and translocations. Thus, our findings implicate a general role for these factors in promoting end joining and thereby preventing DSBs from progressing into chromosomal breaks and translocations. As cellular p53 status does not markedly influence the frequency of such events, our results also have implications for how p53 and the DSB response machinery cooperate to suppress generation of lymphomas with oncogenic translocations.     

Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified

Background  

In recent years, model based approaches such as maximum likelihood have become the methods of choice for constructing phylogenies. A number of authors have shown the importance of using adequate substitution models in order to produce accurate phylogenies. In the past, many empirical models of amino acid substitution have been derived using a variety of different methods and protein datasets. These matrices are normally used as surrogates, rather than deriving the maximum likelihood model from the dataset being examined. With few exceptions, selection between alternative matrices has been carried out in an ad hoc manner.     

Investigation of de novo cholesterol synthetic capacity in the gonads of goldfish (Carassius auratus) exposed to the phytosterol beta-sitosterol

Total and intra-mitochondrial gonadal cholesterol concentrations are decreased in fish exposed to the phytoestrogen beta-sitosterol (beta-sit). The present study examined the potential for beta-sit to disrupt de novo cholesterol synthesis in the gonads of goldfish exposed to 200 microgram/g beta-sit and 10 microgram/g 17beta-estradiol (E2; estrogenic control) by intra-peritoneal Silastic^® implants for 21 days. The de novo cholesterol synthetic capacity was estimated by incubating gonadal tissue with 14C-acetate for a period of 18 hours, followed by chloroform/methanol lipid extraction and thin layer chromatography (TLC) lipid separation. Lipid classes were confirmed using infrared spectroscopy. Plasma testosterone (T) and total cholesterol concentration were measured and gonadosomatic index (GSI) was calculated. Plasma T was significantly reduced in male beta-sit-treated fish compared to control and E2-treated fish (p < 0.001). 14C-Acetate incorporation into cholesterol and cholesterol esters was not significantly different among treatment groups for male and female fish, however, 14C-enrichment was higher than expected in both triglycerides (TG) and free fatty acids (FFA). FFA incorporation was significantly higher in male control fish than either beta-sit or E2 treatments (p = 0.005). Plasma cholesterol concentration was significantly increased in the male beta-sit treatment group compared to controls (p = 0.027). These results indicate gonadal de novo cholesterol biosynthetic capacity is not disrupted by beta-sit or E2 treatment in early recrudescing male or female goldfish, while plasma cholesterol and steroid concentrations are sensitive to beta-sit exposure.     

Assessment of neuromuscular and haemodynamic activity in individuals with and without chronic low back pain

Background

Biering-Sørenson (1984) found that individuals with less lumbar extensor muscle endurance had an increased occurrence of first episode low back pain. As a result, back endurance tests have been recommended for inclusion in health assessment protocols. However, different studies have reported markedly different values for endurance times, leading some researchers to believe that the back is receiving support from the biceps femoris and gluteus maximus. Therefore, this study was designed to examine the haemodynamic and neuromuscular activity of the erector spinae, biceps femoris, and gluteus maximus musculature during the Biering-Sørenson Muscular Endurance Test (BSME).

Methods

Seventeen healthy individuals and 46 individuals with chronic low back pain performed the Biering-Sørenson Muscular Endurance Test while surface electromyography was used to quantify neuromuscular activity. Disposable silver-silver-chloride electrodes were placed in a bipolar arrangement over the right or left biceps femoris, gluteus maximus, and the lumbosacral paraspinal muscles at the level of L₃. Near Infrared Spectroscopy was used simultaneously to measure tissue oxygenation and blood volume changes of the erector spinae and biceps femoris.

Results

The healthy group displayed a significantly longer time to fatigue (Healthy: 168.5s, LBP: 111.1s; p ≤ 0.05). Significant differences were shown in the median frequency slope of the erector spinae between the two groups at 90–100% of the time to fatigue while no significant differences were noted in the haemodynamic data for the two groups.

Conclusion

Although the BSME has been recognized as a test for back endurance, individuals with chronic LBP appear to incorporate a strategy that may help support the back musculature by utilizing the biceps femoris and gluteus maximus to a greater degree than their healthy counterparts.

     

Comparison of variability associated with sample preparation in two-dimensional gel electrophoresis of cardiac tissue.

Variability is a major complicating factor in analysis by two-dimensional gel electrophoresis. Improvements in methodologies have focused on improving individual gel quality rather than reproducibility. We homogenized rat cardiac tissue and rehydrated using a matrix of buffers to determine the optimal sample conditions. Six buffers were used to solubilize the proteins. Solubilized proteins were separated by isoelectric focusing using four buffers. Gels were run in triplicate to assess the method of preparation yielding the least variability. Number of spots and variability were different between conditions. Proteins solubilized in a buffer containing 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, ampholytes, DTT, and protease inhibitors and focused in a buffer containing 9 M urea and 4% NP40 had the lowest coefficient of variation. Variability was compared across isoelectric point ranges and was different. Minimizing technical variability in two-dimensional polyacrylamide gel electrophoresis is critical to identify differences between conditions. Sample preparation should be optimized to minimize variability as well as to maximize the number of spots seen.     

Psychometrics of the Balance Beam Functional Test in C57BL/6 MiceReferences

Aging is associated with a progressive decline in physical function characterized by decreased mobility, which is an important risk factor for loss of independence and reduced quality of life. Functional testing conducted in animals has advanced our understanding of age-related changes in physical ability and contributed to the development of physiologic measurements that can be used to assess functional changes during aging. The balance beam test is one assessment tool used to measure age-related changes in balance and coordination. The goal of this study is to provide analytical examples and psychometric support of a protocol that has been analyzed to show how the number of successive test runs, foot slips, pauses, and hesitations affect the reliability of the primary outcome measure, which is the time to cross the beam. Our results suggest that conducting more than 1 training session, consisting of greater than or equal to 3 successful training runs, followed by at least one test session with no less than 2 successful runs (that is, runs without pauses or hesitations) provides a psychometrically sound outcome. The data presented here indicate that a psychometric approach can improve protocol design and reliability of balance beam measures in mice.

The world’s population is aging. For the first time in history, the number of people who are 65 y of age or older has surpassed the number of children under 5 y of age.²⁶ Current population projections estimate that by 2050, one in 6 people in the world will be 65 y of age or older.²⁶ As this aging of the population intensifies, so does the number of older adults with physical limitations.¹⁹ These limitations in physical function impair the ability to perform activities of daily living, reduce the quality of life, and increase the risk of falling.^9,18 One of the most important causes of reduced physical function is age-related loss of skeletal muscle mass.^27,29,30 Emerging evidence suggests the loss of skeletal muscle mass is not the only contributing factor to the decline in physical performance in older adults.²³ For instance, loss of muscle strength, power, and function, distinct features of aging muscle, are strongly associated with reduced mobility and physical ability in older adults.²³Our current understanding of physical function across the continuum of age-related changes has been largely guided by studies conducted in animal models of aging.^13,17 Rodents (primarily rats and mice) are the most commonly used experimental animals in aging research due to their relatively short developmental period and life span as compared with humans.¹³ Many phenotypes and functional metrics associated with biologic aging are conserved across species.¹ Laboratory-based motor measurements have provided numerous assessments of age-related changes in physical function in rodents.^2,5,10,11 Often referred to as behavioral testing because of cognitive influences on physical ability, these tests measure functional status in rodents by assessing a spectrum of physical performance metrics including balance, coordination, agility, and strength.¹² Of these functional assays, the balance beam test is a well-established method used to assess several parameters of motor capabilities and gait-related activity in mice.^4,15 The balance beam apparatus is a simple construct, requires minimal equipment and is easy to assemble (Figure 1). Test measures typically consist of time to cross the beam and the number of foot slips. Performance on the beam allows an assessment of balance-related motor skills, provides a functional marker of locomotor differences between young and aged animals, and permits comparison of the effects of age, disease, and therapeutic interventions between cohorts with regard to motor function, coordination, and balance.^4,6,24,25,28Open in a separate windowFigure 1.Balance beam apparatus. A wooden beam (12 mm and 6 mm) is suspended 50 cm above the ground. To encourage the mouse to cross the beam to enter a secure location, a safe house with bedding is placed at the exit end of the beam. A net provides a soft landing in case the mouse falls from the beam. The camera at the end of the beam captures any foot slips as the mouse crosses the beam.Over time, the mechanics and experimental protocol of the balance beam test have undergone several modifications. One of the first applications of measuring balance and coordination was performed to characterize the locomotor skills of aged animals.²⁸ Early balance beam experiments tested the ability of the animal to remain on a narrow rod during a single 3-min interval. The test was conducted over 3 consecutive days, with latency to fall as the outcome variable.⁴ More recent versions of the balance beam test employ flat boards or round rods suspended horizontally or inclined. The test requires mice to be trained to traverse the beam without being over-trained, as this can desensitize the innate motivation to escape.¹⁵ Testing is conducted within 1 or 2 d after training or within a period of time during which the mouse is likely to retain memory of the learned task.⁶ Measures obtained from the balance beam include (1) the amount of time required to travel a constant distance on the beam (typically 80 cm), (2) the number of foot slips encountered (captured on video recordings), and, in some cases, (3) the number of pauses or nudges required for the mouse to complete the task. In theory, the quantitative components of the balance beam test are relatively straightforward to measure when the animal completes a successful test run by crossing the beam without pausing midway or hesitating at the starting line or before entering the safe house at the end. However, due to unavoidable complications resulting from pauses and hesitations, which are considered intrinsic features of animal testing using the balance beam, the reliability of the primary outcome measure (that is, time to cross) may be questionable. Although thorough and well-crafted balance beam protocols provide useful instructional models,^4,6,8 a standardized analytical strategy is not available to account for potential performance inconsistencies that inherently arise in animal functional testing.Our goal in this study is to provide a pragmatic, psychometrically based analysis to assess the reliability of measures between one successful as run compared with 2 other successful runs and to demonstrate the impact of pauses, hesitations, and foot slips on time to cross the beam. This analysis may provide a more consistent and standardized use of the balance beam test as a tool to assess balance and coordination in mice.     

Mutant and Overexpression Analysis of a C2H2 Single Zinc Finger Gene of Arabidopsis

The goal of this work was to characterize an Arabidopsis C2H2 single zinc finger gene, named AtZFP11, that is similar to SUPERMAN (SUP) and RABBIT EARS (RBE). No altered phenotype was observed in mutants analyzed that were derived through TILLING nor a T-DNA insertion into the exon of AtZFP11. Plants derived by intercrossing AtZFP11 to SUP and RBE alleles did not display any phenotype that could be attributed to AtZFP11. Transgenic plants constitutively overexpressing the AtZFP11 protein displayed severe abnormalities. Phenotypically normal transgenic plants that contained an ecdysone-inducible promoter system fused to the AtZFP11 gene were recovered. These transgenic plants displayed similar abnormalities once induced with methoxyfenozide. Microarray and RT-PCR analysis of gene expression in plants overexpressing AtZFP11 after induction revealed changes in gene expression of a number of genes involved in auxin, jasmonic acid, and stress responses. These results suggest that AtZFP11 plays a role in regulation of gene expression and may be functionally redundant or that conditions needed to observe phenotypic differences in the mutant plants were not provided in the course of these experiments.     

Expression of clock genes in human subcutaneous and visceral adipose tissues

Disrupted circadian rhythms are associated with obesity and metabolic alterations, but little is known about the participation of peripheral circadian clock machinery in these processes. The aim of the present study was to analyze RNA expression of clock genes in subcutaneous (SAT) and visceral (VAT) adipose tissues of male and female subjects in AM (morning) and PM (afternoon) periods, and its interactions with body mass index (BMI). Ninety-one subjects (41 ± 11 yrs of age) presenting a wide range of BMI (21.4 to 48.6 kg/m(2)) were included. SAT and VAT biopsies were obtained from patients undergoing abdominal surgeries. Clock genes expressions were evaluated by qRT-PCR. The only clock gene that showed higher expression (p 相似文献