A model for transition of 5 '-nuclease domain of DNA polymerase I from inert to active modes

Bacteria contain DNA polymerase I (PolI), a single polypeptide chain consisting of similar to 930 residues, possessing DNA-dependent DNA polymerase, 3'-5' proofreading and 5'-3' exonuclease (also known as flap endonuclease) activities. PolI is particularly important in the processing of Okazaki fragments generated during lagging strand replication and must ultimately produce a double-stranded substrate with a nick suitable for DNA ligase to seal. PolI's activities must be highly coordinated both temporally and spatially otherwise uncontrolled 5'-nuclease activity could attack a nick and produce extended gaps leading to potentially lethal double-strand breaks. To investigate the mechanism of how PolI efficiently produces these nicks, we present theoretical studies on the dynamics of two possible scenarios or models. In one the flap DNA substrate can transit from the polymerase active site to the 5'-nuclease active site, with the relative position of the two active sites being kept fixed; while the other is that the 5'-nuclease domain can transit from the inactive mode, with the 5'-nuclease active site distant from the cleavage site on the DNA substrate, to the active mode, where the active site and substrate cleavage site are juxtaposed. The theoretical results based on the former scenario are inconsistent with the available experimental data that indicated that the majority of 5'-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus. By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data. We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5'-3' exonuclease activities. Moreover, predicted results for the latter model are presented

Transkingdom Networks: A Systems Biology Approach to Identify Causal Members of Host-Microbiota Interactions

Improvements in sequencing technologies and reduced experimental costs have resulted in a vast number of studies generating high-throughput data. Although the number of methods to analyze these "omics" data has also increased, computational complexity and lack of documentation hinder researchers from analyzing their high-throughput data to its true potential. In this chapter we detail our data-driven, transkingdom network (TransNet) analysis protocol to integrate and interrogate multi-omics data. This systems biology approach has allowed us to successfully identify important causal relationships between different taxonomic kingdoms (e.g. mammals and microbes) using diverse types of data

Labrador retrievers under primary veterinary care in the UK: demography, mortality and disorders

Abstract Background Labrador retrievers are reportedly predisposed to many disorders but accurate prevalence information relating to the general population are lacking. This study aimed to describe demography, mortality and commonly recorded diseases in Labrador retrievers under UK veterinary care. Methods The VetCompass™ programme collects electronic patient record data on dogs attending UK primary-care veterinary practices. Demographic analysis covered all33,320 Labrador retrievers in the VetCompass™ database under veterinary care during 2013 while disorder and mortality data were extracted from a random sample of 2074 (6.2%) of these dogs. Results Of the Labrador retrievers with information available, 15,427 (46.4%) were female and 15,252 (53.6%) were male. Females were more likely to be neutered than males (59.7% versus 54.8%, P <  0.001). The overall mean adult bodyweight was 33.0 kg (SD 6.1). Adult males were heavier (35.2 kg, SD 5.9 kg) than adult females (30.4 kg, SD 5.2 kg) (P <  0.001). The median longevity of Labrador retrievers overall was 12.0 years (IQR 9.9–13.8, range 0.0–16.0). The most common recorded colours were black (44.6%), yellow (27.8%) and liver/chocolate (reported from hereon as chocolate) (23.8%). The median longevity of non-chocolate coloured dogs (n = 139, 12.1 years, IQR 10.2–13.9, range 0.0–16.0) was longer than for chocolate coloured animals (n = 34, 10.7 years, IQR 9.0–12.4, range 3.8–15.5) (P = 0.028). Of a random sample of 2074 (6.2%) Labrador retrievers under care in 2013 that had full disorder data extracted, 1277 (61.6%) had at least one disorder recorded. The total number of dogs who died at any date during the study was 176. The most prevalent disorders recorded were otitis externa (n = 215, prevalence 10.4%, 95% CI: 9.1–11.8), overweight/obesity (183, 8.8%, 95% CI: 7.6–10.1) and degenerative joint disease (115, 5.5%, 95% CI: 4.6–6.6). Overweight/obesity was not statistically significantly associated with neutering in females (8.3% of entire versus 12.5% of neutered, P = 0.065) but was associated with neutering in males (4.1% of entire versus 11.4% of neutered, P < 0.001). The prevalence of otitis externa in black dogs was 12.8%, in yellow dogs it was 17.0% but, in chocolate dogs, it rose to 23.4% (P < 0.001). Similarly, the prevalence of pyo-traumatic dermatitis in black dogs was 1.1%, in yellow dogs it was 1.6% but in chocolate dogs it rose to 4.0% (P = 0.011). Conclusions The current study assists prioritisation of health issues within Labrador retrievers. The most common disorders were overweight/obesity, otitis externa and degenerative joint disease. Males were significantly heavier females. These results can alert prospective owners to potential health issues and inform breed-specific wellness checks

Parametric study of EEG sensitivity to phase noise during face processing

&lt;b&gt;Background: &lt;/b&gt; The present paper examines the visual processing speed of complex objects, here faces, by mapping the relationship between object physical properties and single-trial brain responses. Measuring visual processing speed is challenging because uncontrolled physical differences that co-vary with object categories might affect brain measurements, thus biasing our speed estimates. Recently, we demonstrated that early event-related potential (ERP) differences between faces and objects are preserved even when images differ only in phase information, and amplitude spectra are equated across image categories. Here, we use a parametric design to study how early ERP to faces are shaped by phase information. Subjects performed a two-alternative force choice discrimination between two faces (Experiment 1) or textures (two control experiments). All stimuli had the same amplitude spectrum and were presented at 11 phase noise levels, varying from 0% to 100% in 10% increments, using a linear phase interpolation technique. Single-trial ERP data from each subject were analysed using a multiple linear regression model. &lt;b&gt;Results: &lt;/b&gt; Our results show that sensitivity to phase noise in faces emerges progressively in a short time window between the P1 and the N170 ERP visual components. The sensitivity to phase noise starts at about 120–130 ms after stimulus onset and continues for another 25–40 ms. This result was robust both within and across subjects. A control experiment using pink noise textures, which had the same second-order statistics as the faces used in Experiment 1, demonstrated that the sensitivity to phase noise observed for faces cannot be explained by the presence of global image structure alone. A second control experiment used wavelet textures that were matched to the face stimuli in terms of second- and higher-order image statistics. Results from this experiment suggest that higher-order statistics of faces are necessary but not sufficient to obtain the sensitivity to phase noise function observed in response to faces. &lt;b&gt;Conclusion: &lt;/b&gt; Our results constitute the first quantitative assessment of the time course of phase information processing by the human visual brain. We interpret our results in a framework that focuses on image statistics and single-trial analyses

A randomised trial and economic evaluation of the effect of response mode on response rate, response bias, and item non-response in a survey of doctors

<p>Abstract</p> <p>Background</p> <p>Surveys of doctors are an important data collection method in health services research. Ways to improve response rates, minimise survey response bias and item non-response, within a given budget, have not previously been addressed in the same study. The aim of this paper is to compare the effects and costs of three different modes of survey administration in a national survey of doctors.</p> <p>Methods</p> <p>A stratified random sample of 4.9% (2,702/54,160) of doctors undertaking clinical practice was drawn from a national directory of all doctors in Australia. Stratification was by four doctor types: general practitioners, specialists, specialists-in-training, and hospital non-specialists, and by six rural/remote categories. A three-arm parallel trial design with equal randomisation across arms was used. Doctors were randomly allocated to: online questionnaire (902); simultaneous mixed mode (a paper questionnaire and login details sent together) (900); or, sequential mixed mode (online followed by a paper questionnaire with the reminder) (900). Analysis was by intention to treat, as within each primary mode, doctors could choose either paper or online. Primary outcome measures were response rate, survey response bias, item non-response, and cost.</p> <p>Results</p> <p>The online mode had a response rate 12.95%, followed by the simultaneous mixed mode with 19.7%, and the sequential mixed mode with 20.7%. After adjusting for observed differences between the groups, the online mode had a 7 percentage point lower response rate compared to the simultaneous mixed mode, and a 7.7 percentage point lower response rate compared to sequential mixed mode. The difference in response rate between the sequential and simultaneous modes was not statistically significant. Both mixed modes showed evidence of response bias, whilst the characteristics of online respondents were similar to the population. However, the online mode had a higher rate of item non-response compared to both mixed modes. The total cost of the online survey was 38% lower than simultaneous mixed mode and 22% lower than sequential mixed mode. The cost of the sequential mixed mode was 14% lower than simultaneous mixed mode. Compared to the online mode, the sequential mixed mode was the most cost-effective, although exhibiting some evidence of response bias.</p> <p>Conclusions</p> <p>Decisions on which survey mode to use depend on response rates, response bias, item non-response and costs. The sequential mixed mode appears to be the most cost-effective mode of survey administration for surveys of the population of doctors, if one is prepared to accept a degree of response bias. Online surveys are not yet suitable to be used exclusively for surveys of the doctor population.</p

PKCδ regulates force signaling during VEGF/CXCL4 induced dissociation of endothelial tubes

Wound healing requires the vasculature to re-establish itself from the severed ends; endothelial cells within capillaries must detach from neighboring cells before they can migrate into the nascent wound bed to initiate angiogenesis. The dissociation of these endothelial capillaries is driven partially by platelets' release of growth factors and cytokines, particularly the chemokine CXCL4/platelet factor-4 (PF4) that increases cell-cell de-adherence. As this retraction is partly mediated by increased transcellular contractility, the protein kinase c-δ/myosin light chain-2 (PKCδ/MLC-2) signaling axis becomes a candidate mechanism to drive endothelial dissociation. We hypothesize that PKCδ activation induces contractility through MLC-2 to promote dissociation of endothelial cords after exposure to platelet-released CXCL4 and VEGF. To investigate this mechanism of contractility, endothelial cells were allowed to form cords following CXCL4 addition to perpetuate cord dissociation. In this study, CXCL4-induced dissociation was reduced by a VEGFR inhibitor (sunitinib malate) and/or PKCδ inhibition. During combined CXCL4+VEGF treatment, increased contractility mediated by MLC-2 that is dependent on PKCδ regulation. As cellular force is transmitted to focal adhesions, zyxin, a focal adhesion protein that is mechano-responsive, was upregulated after PKCδ inhibition. This study suggests that growth factor regulation of PKCδ may be involved in CXCL4-mediated dissociation of endothelial cords. © 2014 Jamison et al

Increased Infarct Wall Thickness by a Bio-Inert Material Is Insufficient to Prevent Negative Left Ventricular Remodeling after Myocardial Infarction

Several injectable materials have been shown to preserve or improve cardiac function as well as prevent or slow left ventricular (LV) remodeling post-myocardial infarction (MI). However, it is unclear as to whether it is the structural support or the bioactivity of these polymers that lead to beneficial effects. Herein, we examine how passive structural enhancement of the LV wall by an increase in wall thickness affects cardiac function post-MI using a bio-inert, non-degradable synthetic polymer in an effort to better understand the mechanisms by which injectable materials affect LV remodeling.Poly(ethylene glycol) (PEG) gels of storage modulus G' = 0.5±0.1 kPa were injected and polymerized in situ one week after total occlusion of the left coronary artery in female Sprague Dawley rats. The animals were imaged using magnetic resonance imaging (MRI) at 7±1 day(s) post-MI as a baseline and again post-injection 49±4 days after MI. Infarct wall thickness was statistically increased in PEG gel injected vs. control animals (p<0.01). However, animals in the polymer and control groups showed decreases in cardiac function in terms of end diastolic volume, end systolic volume and ejection fraction compared to baseline (p<0.01). The cellular response to injection was also similar in both groups.The results of this study demonstrate that passive structural reinforcement alone was insufficient to prevent post-MI remodeling, suggesting that bioactivity and/or cell infiltration due to degradation of injectable materials are likely playing a key role in the preservation of cardiac function, thus providing a deeper understanding of the influencing properties of biomaterials necessary to prevent post-MI negative remodeling

Electrocautery causes more ischemic peritoneal tissue damage than ultrasonic dissection

Contains fulltext : 96869.pdf (publisher's version ) (Open Access)BACKGROUND: Minimizing peritoneal tissue injury during abdominal surgery has the benefit of reducing postoperative inflammatory response, pain, and adhesion formation. Ultrasonic dissection seems to reduce tissue damage. This study aimed to compare electrocautery and ultrasonic dissection in terms of peritoneal tissue ischemia measured by microdialysis. METHODS: In this study, 18 Wistar rats underwent a median laparotomy and had a peritoneal microdialysis catheter implanted in the left lateral sidewall. The animals were randomly assigned to receive two standard peritoneal incisions parallel to the catheter by either ultrasonic dissection or electrocautery. After the operation, samples of microdialysis dialysate were taken every 2 h until 72 h postoperatively for measurements of pyruvate, lactate, glucose, and glycerol, and ratios were calculated. RESULTS: The mean lactate-pyruvate ratio (LPR), lactate-glucose ratio (LGR), and glycerol concentration were significantly higher in the electrocautery group than in the ultrasonic dissection group until respectively 34, 48, and 48 h after surgery. The mean areas under the curve (AUC) of LPR, LGR, and glycerol concentration also were higher in the electrocautery group than in the ultrasonic dissection group (4,387 vs. 1,639, P=0.011; 59 vs. 21, P=0.008; 7,438 vs. 4,169, P=0.008, respectively). CONCLUSION: Electrosurgery causes more ischemic peritoneal tissue damage than ultrasonic dissection.01 juni 201

Action Mechanism of Inhibin α-Subunit on the Development of Sertoli Cells and First Wave of Spermatogenesis in Mice

Inhibin is an important marker of Sertoli cell (SC) activity in animals with impaired spermatogenesis. However, the precise relationship between inhibin and SC activity is unknown. To investigate this relationship, we partially silenced both the transcription and translation of the gene for the α-subunit of inhibin, Inha, using recombinant pshRNA vectors developed with RNAi-Ready pSIREN-RetroQ-ZsGreen Vector (Clontech Laboratories, Mountain View, Calif). We found that Inha silencing suppresses the cell-cycle regulators Cyclin D1 and Cyclin E and up-regulates the cell-cycle inhibitor P21 (as detected by Western blot analysis), thereby increasing the number of SCs in the G1 phase of the cell cycle and decreasing the amount in the S-phase of the cell cycle (as detected by flow cytometry). Inha silencing also suppressed Pdgfa, Igf1, and Kitl mRNA levels and up-regulated Tgfbrs, Inhba, Inhbb, Cyp11a1, Dhh, and Tjp1 mRNA levels (as indicated by real-time polymerase chain reaction [PCR] analysis). These findings indicate that Inha has the potential to influence the availability of the ligand inhibin and its antagonist activin in the SC in an autocrine manner and inhibit the progression of SC from G1 to S. It may also participate in the development of the blood–testis barrier, Leydig cells, and spermatogenesis through its effect on Dhh, Tjp1, Kitl, and Pdgfa. Real-time PCR and Western blot analyses of Inha, Inhba, and Inhbb mRNA and Inha levels over time show that Inha plays an important role in the formation of round spermatid during the first wave of spermatogenesis in mice