DNA double-strand breaks (DSBs) threaten genomic stability, since their persistence can lead to loss of critical genetic information, chromosomal translocations or rearrangements, and cell death. ...DSBs can be repaired through the nonhomologous end-joining pathway (NHEJ), which processes and ligates DNA ends efficiently to prevent or minimize sequence loss. Polymerase λ (Polλ), one of the Family X polymerases, fills sequence gaps of DSB substrates with a strict specificity for a base-paired primer terminus. There is little information regarding Polλ's approach to engaging such substrates. We used in vitro polymerization and cell-based NHEJ assays to explore the contributions of conserved loop regions toward DSB substrate specificity and utilization. In addition, we present multiple crystal structures of Polλ in synapsis with varying biologically relevant DSB end configurations, revealing how key structural features and hydrogen bonding networks work in concert to stabilize these tenuous, potentially cytotoxic DNA lesions during NHEJ.
DNA ligase IV (LigIV) performs the final DNA nick-sealing step of classical nonhomologous end-joining, which is critical for immunoglobulin gene maturation and efficient repair of genotoxic DNA ...double-strand breaks. Hypomorphic LigIV mutations cause extreme radiation sensitivity and immunodeficiency in humans. To better understand the unique features of LigIV function, here we report the crystal structure of the catalytic core of human LigIV in complex with a nicked nucleic acid substrate in two distinct states-an open lysyl-AMP intermediate, and a closed DNA-adenylate form. Results from structural and mutagenesis experiments unveil a dynamic LigIV DNA encirclement mechanism characterized by extensive interdomain interactions and active site phosphoanhydride coordination, all of which are required for efficient DNA nick sealing. These studies provide a scaffold for defining impacts of LigIV catalytic core mutations and deficiencies in human LIG4 syndrome.
DNA polymerase (pol) μ is a DNA-dependent polymerase that incorporates nucleotides during gap-filling synthesis in the non-homologous end-joining pathway of double-strand break repair. Here we report ...time-lapse X-ray crystallography snapshots of catalytic events during gap-filling DNA synthesis by pol μ. Unique catalytic intermediates and active site conformational changes that underlie catalysis are uncovered, and a transient third (product) metal ion is observed in the product state. The product manganese coordinates phosphate oxygens of the inserted nucleotide and PP
. The product metal is not observed during DNA synthesis in the presence of magnesium. Kinetic analyses indicate that manganese increases the rate constant for deoxynucleoside 5'-triphosphate insertion compared to magnesium. The likely product stabilization role of the manganese product metal in pol μ is discussed. These observations provide insight on structural attributes of this X-family double-strand break repair polymerase that impact its biological function in genome maintenance.DNA polymerase (pol) μ functions in DNA double-strand break repair. Here the authors use time-lapse X-ray crystallography to capture the states of pol µ during the conversion from pre-catalytic to product complex and observe a third transiently bound metal ion in the product state.
1. The effects of harvest on the annual and seasonal survival of willow ptarmigan Lagopus lagopus L. were tested in a large-scale harvest experiment. Management units were randomly assigned to one of ...three experimental treatments: 0%, 15% or 30% harvest. Seasonal quotas were based on the experimental treatment and estimates of bird density before the hunting season. Survival rates and hazard functions for radio-marked ptarmigan were then estimated under the competing risks of harvest and natural mortality. 2. The partially compensatory mortality hypothesis was supported: annual survival of ptarmigan was 0·54 ± 0·08 SE under 0% harvest, 0·47 ± 0·06 under 15% harvest, and was reduced to 0·30 ± 0·05 under 30% harvest. Harvest mortality increased linearly from 0·08 ± 0·05, 0·27 ± 0·05 and 0·42 ± 0·06 from 0% to 30% harvest, whereas natural mortality was 0·38 ± 0·08, 0·25 ± 0·05 and 0·28 ± 0·06 under the same treatments. 3. Realized risk of harvest mortality was 0·08-0·12 points higher than our set harvest treatments of 0-30% because birds were exposed to risk if they moved out of protected areas. The superadditive hypothesis was supported because birds in the 30% harvest treatment had higher natural mortality during winter after the hunting season. 4. Natural mortality was mainly because of raptor predation, with two seasonal peaks in fall and spring. Natural and harvest mortality coincided during early autumn with little potential for compensation during winter months. Peak risk of harvest mortality was 5× higher than natural mortality. Low natural mortality during winter suggests that most late season harvest would be additive mortality. 5. Environmental correlates of natural mortality of ptarmigan included seasonal changes in snow cover, onset of juvenile dispersal, and periods of territorial activity. Natural mortality of ptarmigan was highest during autumn movements and nesting by gyrfalcons Falco rusticolus L. Mortality was low when gyrfalcons had departed for coastal wintering sites, and during summer when ptarmigan were attending nests and broods. 6. Our experimental results have important implications for harvest management of upland gamebirds. Seasonal quotas based on proportional harvest were effective and should be set at ≤15% of August populations for regional management plans. Under threshold harvest of a reproductive surplus, 15% harvest would be sustainable at productivity rates ≥2·5 young per pair. Impacts of winter harvest could be minimized by closing the hunting season in early November or by reducing late season quotas.
Echolocating greater horseshoe bats (Rhinolophus ferrumequinum) emit their biosonar pulses nasally, through nostrils surrounded by fleshy appendages ('noseleaves') that diffract the outgoing ...ultrasonic waves. Movements of one noseleaf part, the lancet, were measured in live bats using two synchronized high speed video cameras with 3D stereo reconstruction, and synchronized with pulse emissions recorded by an ultrasonic microphone. During individual broadcasts, the lancet briefly flicks forward (flexion) and is then restored to its original position. This forward motion lasts tens of milliseconds and increases the curvature of the affected noseleaf surfaces. Approximately 90% of the maximum displacements occurred within the duration of individual pulses, with 70% occurring towards the end. Similar lancet motions were not observed between individual pulses in a sequence of broadcasts. Velocities of the lancet motion were too small to induce Doppler shifts of a biologically-meaningful magnitude, but the maximum displacements were significant in comparison with the overall size of the lancet and the ultrasonic wavelengths. Three finite element models were made from micro-CT scans of the noseleaf post mortem to investigate the acoustic effects of lancet displacement. The broadcast beam shapes were found to be altered substantially by the observed small lancet movements. These findings demonstrate that-in addition to the previously described motions of the anterior leaf and the pinna-horseshoe bat biosonar has a third degree of freedom for fast changes that can happen on the time scale of the emitted pulses or the returning echoes and could provide a dynamic mechanism for the encoding of sensory information.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Heparan sulfate (HS) is present on the surface of endothelial and surrounding tissues in large quantities. It plays important roles in regulating numerous functions of the blood vessel wall, ...including blood coagulation, inflammation response, and cell differentiation. HS is a highly sulfated polysaccharide containing glucosamine and glucuronic/iduronic acid repeating disaccharide units. The unique sulfated saccharide sequences of HS determine its specific functions. Heparin, an analog of HS, is the most commonly used anticoagulant drug. Because of its wide range of biological functions, HS has become an interesting molecule to biochemists, medicinal chemists, and developmental biologists. In this review, we summarize recent progress toward understanding the interaction between HS and blood-coagulating factors, the biosynthesis of anticoagulant HS and the mechanism of action of HS biosynthetic enzymes. Furthermore, knowledge of the biosynthesis of HS facilitates the development of novel enzymatic approaches to synthesize HS from bacterial capsular polysaccharides and to produce polysaccharide end products with high specificity for the biological target. These advancements provide the foundation for the development of polysaccharide-based therapeutic agents.
Summary Background Vitamin K antagonists, the current standard treatment for prophylaxis against stroke and systemic embolism in patients with atrial fibrillation, require regular monitoring and dose ...adjustment; an unmonitored, fixed-dose anticoagulant regimen would be preferable. The aim of this randomised, open-label non-inferiority trial was to compare the efficacy and safety of idraparinux with vitamin K antagonists. Methods Patients with atrial fibrillation at risk for thromboembolism were randomly assigned to receive either subcutaneous idraparinux (2·5 mg weekly) or adjusted-dose vitamin K antagonists (target of an international normalised ratio of 2–3). Assessment of outcome was done blinded to treatment. The primary efficacy outcome was the cumulative incidence of all stroke and systemic embolism. The principal safety outcome was clinically relevant bleeding. Analyses were done by intention to treat; the non-inferiority hazard ratio was set at 1·5. This trial is registered with ClinicalTrials.gov , number NCT00070655. Findings The trial was stopped after randomisation of 4576 patients (2283 to receive idraparinux, 2293 to receive vitamin K antagonists) and a mean follow-up period of 10·7 (SD 5·4) months because of excess clinically relevant bleeding with idraparinux (346 cases vs 226 cases; 19·7 vs 11·3 per 100 patient-years; p<0·0001). There were 21 instances of intracranial bleeding with idraparinux and nine with vitamin K antagonists (1·1 vs 0·4 per 100 patient-years; p=0·014); elderly patients and those with renal impairment were at greater risk of such complications. There were 18 cases of thromboembolism with idraparinux and 27 cases with vitamin K antagonists (0·9 vs 1·3 per 100 patient-years; hazard ratio 0·71, 95% CI 0·39–1·30; p=0·007), satisfying the non-inferiority criterion. There were 62 deaths with idraparinux and 61 with vitamin K anatagonists (3·2 vs 2·9 per 100 patient-years; p=0·49). Interpretation In patients with atrial fibrillation at risk for thromboembolism, long-term treatment with idraparinux was no worse than vitamin K antagonists in terms of efficacy, but caused significantly more bleeding.
Background Sensitization to cockroach allergens is a major risk factor for asthma. The cockroach allergen Bla g 1 has multiple repeats of approximately 100 amino acids, but the fold of the protein ...and its biological function are unknown. Objective We sought to determine the structure of Bla g 1, investigate the implications for allergic disease, and standardize cockroach exposure assays. Methods nBla g 1 and recombinant constructs were compared by using ELISA with specific murine IgG and human IgE. The structure of Bla g 1 was determined by x-ray crystallography. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to examine the ligand-binding properties of the allergen. Results The structure of an rBla g 1 construct with comparable IgE and IgG reactivity to the natural allergen was solved by x-ray crystallography. The Bla g 1 repeat forms a novel fold with 6 helices. Two repeats encapsulate a large and nearly spherical hydrophobic cavity, defining the basic structural unit. Lipids in the cavity varied depending on the allergen origin. Palmitic, oleic, and stearic acids were associated with nBla g 1 from cockroach frass. One unit of Bla g 1 was equivalent to 104 ng of allergen. Conclusions Bla g 1 has a novel fold with a capacity to bind various lipids, which suggests a digestive function associated with nonspecific transport of lipid molecules in cockroaches. Defining the basic structural unit of Bla g 1 facilitates the standardization of assays in absolute units for the assessment of environmental allergen exposure.
Abstract
Background
Associations of birthweight, childhood body size and pubertal timing with breast cancer risks by menopausal status and tumor receptor subtypes are inconclusive. Thus, we ...investigated these associations in a population-based cohort of Danish women.
Methods
We studied 162,419 women born between 1930 and 1996 from the Copenhagen School Health Records Register. The register includes information on birthweight, measured childhood weights and heights at the age of 7–13 years, and computed ages at the onset of the growth spurt (OGS) and at peak height velocity (PHV). The Danish Breast Cancer Cooperative Group database provided information on breast cancer (
n
= 7510), including estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2) and menopausal status. Hormone replacement therapy use came from the Danish National Prescription Registry. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated by Cox regression.
Results
We found that birthweight was not associated with any breast cancer subtypes. While childhood BMI was not statistically significantly associated with ER+ tumors nor consistently with ER− tumors among pre-menopausal women, consistent inverse associations were found among postmenopausal women. At the age of 7 years, the HRs for postmenopausal ER+ and ER− tumors were 0.90 (95% CI 0.87–0.93) and 0.84 (95% CI 0.79–0.91) per BMI
z
-score, respectively. Similarly, childhood BMI was inversely associated with pre- and postmenopausal HER2− tumors, but not with HER2+ tumors. Childhood height was positively associated with both pre- and postmenopausal ER+ tumors, but not with ER− tumors. At the age of 7 years, the HRs for postmenopausal ER+ and ER− tumors were 1.09 (95% CI 1.06–1.12) and 1.02 (95% CI 0.96–1.09) per height
z
-score, respectively. In general, childhood height was positively associated with HER2+ and HER2− tumors among pre- and postmenopausal women. Ages at OGS and PHV were not associated with any breast cancer subtypes.
Conclusions
We showed that a high BMI and short stature in childhood are associated with reduced risks of certain breast cancer subtypes. Thus, childhood body composition may play a role in the development of breast cancer.
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