The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses ...for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.
► Tillage selects species according to size, life forms, seed weight and dispersal. ► Herbicides filter species according to their germination and flowering phenology. ► Species on the increase have ...a large germination and flowering range.
Two large-scale weed surveys conducted on winter wheat crops in France in the 1970s and the 2000s were used to determine the influence of management on weed communities. A trait-based approach was used to identify the mechanisms associated with the changing status of arable weeds over a 30 year period.
A three-table ordination method (RLQ analysis) of the data set was performed to relate the environmental table to the species trait table using a species composition table to extract the joint structure (synchronic analysis). We then conducted a diachronic analysis to investigate the relationship between traits, alone or in combination, and the changing status of weeds.
The synchronic analysis showed that tillage intensity filtered weeds according to height, seed weight, life forms and dispersal. Conversely, herbicides selected for species with delayed germination, which allows them to escape herbicide treatments. The diachronic analysis showed that successful weeds that have expanded were small plants, with rather light seeds, that can germinate over a long time frame during the vegetative period. This trait syndrome was probably favoured by profound changes in crop rotation and by increasing herbicide pressure. Our approach provides an excellent example of how future shifts in weed communities can be predicted and hence how weed management can be adapted so as to avoid promoting selection of problematic weeds.
Background
Enhanced recovery pathways (ERPs) aim to improve patient recovery. However, validated outcome measures to evaluate this complex process are lacking. The objective of this review was to ...identify how recovery is measured in ERP studies and to provide recommendations for the design of future studies.
Methods
A systematic search of MEDLINE, Embase and Cochrane databases was conducted. Prospective studies evaluating ERPs compared with traditional care in abdominal surgery published between 2000 and 2013 were included. All reported outcomes were classified into categories: biological and physiological variables, symptom status, functional status, general health perceptions and quality of life (QoL). The phase of recovery measured was defined as baseline, intermediate (in hospital) and late (following discharge).
Results
A total of 38 studies were included based on the systematic review criteria. Biological or physiological variables other than postoperative complications were reported in 30 studies, and included return of gastrointestinal function (25 studies), pulmonary function (5) and physical strength (3). Patient‐reported symptoms, including pain (16 studies) and fatigue (9), were reported less commonly. Reporting of functional status outcomes, including mobilization (16 studies) and ability to perform activities of daily living (4), was similarly uncommon. Health aspects of QoL were reported in only seven studies. Length of follow‐up was generally short, with 24 studies reporting outcomes within 30 days or less. All studies documented in‐hospital outcomes (intermediate phase), but only 17 reported postdischarge outcomes (late phase) other than complications or readmission.
Conclusion
Patient‐reported outcomes, particularly postdischarge functional status, were not commonly reported. Future studies of the effectiveness of ERPs should include validated, patient‐reported outcomes to estimate better their impact on recovery, particularly after discharge from hospital.
Many ways to define recovery
Poly (ADP)-ribose polymerase 1 (PARP1) is an abundant nuclear protein well-known for its role in DNA repair yet also participates in DNA replication, transcription, and co-transcriptional splicing, ...where DNA is undamaged. Thus, binding to undamaged regions in DNA and RNA is likely a part of PARP1's normal repertoire. Here we describe analyses of PARP1 binding to two short single-stranded DNAs, a single-stranded RNA, and a double stranded DNA. The investigations involved comparing the wild-type (WT) full-length enzyme with mutants lacking the catalytic domain (∆CAT) or zinc fingers 1 and 2 (∆Zn1∆Zn2). All three protein types exhibited monomeric characteristics in solution and formed saturated 2:1 complexes with single-stranded T
and U
oligonucleotides. These complexes formed without accumulation of 1:1 intermediates, a pattern suggestive of positive binding cooperativity. The retention of binding activities by ∆CAT and ∆Zn1∆Zn2 enzymes suggests that neither the catalytic domain nor zinc fingers 1 and 2 are indispensable for cooperative binding. In contrast, when a double stranded 19mer DNA was tested, WT PARP1 formed a 4:1 complex while the ∆Zn1Zn2 mutant binding saturated at 1:1 stoichiometry. These deviations from the 2:1 pattern observed with T
and U
oligonucleotides show that PARP's binding mechanism can be influenced by the secondary structure of the nucleic acid. Our studies show that PARP1:nucleic acid interactions are strongly dependent on the nucleic acid type and properties, perhaps reflecting PARP1's ability to respond differently to different nucleic acid ligands in cells. These findings lay a platform for understanding how the functionally versatile PARP1 recognizes diverse oligonucleotides within the realms of chromatin and RNA biology.
Background
Speaking invitations are used by faculty promotion committees as evidence of external recognition. However, women are underrepresented as speakers at specialty society conferences despite ...the rise in women physicians. The purpose of this study was to estimate to what extent the gender of session conveners is associated with the gender distribution of invited speakers at SAGES meetings.
Methods
A retrospective audit of annual SAGES meeting programs during 2009–2018 was performed. All invited panel speakers, defined as faculty delivering a prepared oral presentation in a session under the organization of one or more chairs, were identified. The gender of speakers and chairs/co-chairs was determined. Hands-on courses, paper sessions, military symposia, mock trials, and jeopardy sessions were excluded. We compared the proportion of all-male panels in sessions with all-male conveners versus sessions with at least one woman convener. Statistical analysis was performed using Chi-square and
t
tests.
Results
There were 3405 speakers and 459 panels identified. After applying inclusion and exclusion criteria, 2836 invited speakers on 402 panels were analyzed. Women represented 15% of all speakers, increasing from 9 to 19% (2009 to 2018). This reflects the rise in the proportion of women overall members (11% in 2010 to 19% in 2018). The proportion of panels with at least one woman convener increased from 12 to 58%. All-male panels represented 40% of all panels (
n
= 163) and their proportion significantly decreased over time from 50 to 31% (
p
trend < 0.000). Sessions with all-male conveners had 52% all-male panels, while sessions with at least one woman convener had 19% all-male panels (
p
< 0.001).
Conclusion
The proportion of women invited speakers at the annual SAGES meeting has significantly increased over time. All-male convener sessions were more likely to convene all-male speaker panels. Including a woman chair/co-chair increased the number of women speakers and is a successful strategy to achieve gender balance in conference planning.
Graphic abstract
Summary
We measured the distance 112 patients walked in 6 min, as well as their peak oxygen consumption pedalling a bicycle, week before scheduled resection of benign or malignant colorectal disease. ...The distance walked correlated with peak oxygen consumption, the former ‘accounting’ for about half the variation in the latter, r2 0.52 (95% CI 0.38–0.64), p < 0.0001. In the first postoperative month, 42/112 patients experienced a complication. In multivariate analysis, complications were less likely with longer walking distances and increasing age: the odds ratio (95% CI) reduced to 0.995 (0.990–0.999) for each metre distance, and to 0.96 (0.93–0.99) with each year of age, p = 0.025 and p = 0.018, respectively. The distance walked in 6 min before surgery can provide prognostic information when cardiopulmonary exercise testing is unavailable.
This article is accompanied by an Editorial by Carlisle, pp 799–803 of this issue.
This paper presents a microrobot of continuum type that satisfies constraints in terms of degrees of freedom, size, and safety for endovascular surgery. The proposed instrument, which integrates ...multiple microrobots and a catheter, is called the Multi Active LInk CAtheter (MALICA). Its structure is redundant, compliant, and compact, which makes it a well-suited solution to control the catheter shape. In this paper, we focus on a single microrobot and its actuation to control the orientation of the catheter's distal extremity. The kinematic and differential kinematic models of the microrobot are developed. Because of its kinematic redundancy, the orientation control is formulated as an optimization problem. A differential model-based control scheme is proposed and experimentally validated.
The McGill Inanimate System for Training and Evaluation of Laparoscopic Skills (MISTELS) is a series of five tasks with an objective scoring system. The purpose of this study was to estimate the ...interrater and test-retest reliability of the MISTELS metrics and to assess their internal consistency.
To determine interrater reliability, two trained observers scored 10 subjects, either live or on tape. Test-retest reliability was assessed by having 12 subjects perform two tests, the second immediately following the first. Interrater and test-retest reliability were assessed using intraclass correlation coefficients. Internal consistency between tasks was estimated using Cronbach's alpha.
The interrater and test-retest reliabilities for the total scores were both excellent at 0.998 95% confidence interval (CI), 0.985-1.00 and 0.892 (95% CI, 0.665-0.968), respectively. Cronbach's alpha for the first assessment of the test-retest was 0.86.
The MISTELS metrics have excellent reliability, which exceeds the threshold level of 0.8 required for high-stakes evaluations. These findings support the use of MISTELS for evaluation in many different settings, including residency training programs.
The Munc13 family of exocytosis regulators has multiple Ca(2+)-binding, C2 domains. Here, we probed the mechanism by which Munc13-4 regulates in vitro membrane fusion and platelet exocytosis. We show ...that Munc13-4 enhances in vitro soluble NSF attachment protein receptor (SNARE)-dependent, proteoliposome fusion in a Ca(2+)- and phosphatidylserine (PS)-dependent manner that was independent of SNARE concentrations. Munc13-4-SNARE interactions, under the conditions used, were minimal in the absence or presence of Ca(2+). However, Munc13-4 was able to bind and cluster liposomes harbouring PS in response to Ca(2+). Interestingly, Ca(2+)-dependent liposome binding/clustering and enhancement of proteoliposome fusion required both Munc13-4 C2 domains, but only the Ca(2+)-liganding aspartate residues of the C2B domain. Analytical ultracentrifugation (AUC) measurements indicated that, in solution, Munc13-4 was a monomeric prolate ellipsoid with dimensions consistent with a molecule that could bridge two fusing membranes. To address the potential role of Munc13-4 as a tethering protein in platelets, we examined mepacrine-stained, dense granule mobility and secretion in platelets from wild-type and Munc13-4 null (Unc13d(Jinx)) mice. In the absence of Munc13-4, dense granules were highly mobile in both resting and stimulated platelets, and stimulation-dependent granule release was absent. These observations suggest that dense granules are stably docked in resting platelets awaiting stimulation and that Munc13-4 plays a vesicle-stabilizing or tethering role in resting platelets and also in activated platelets in response to Ca(2+). In summary, we show that Munc13-4 conveys Ca(2+) sensitivity to platelet SNARE-mediated membrane fusion and reveal a potential mechanism by which Munc13-4 bridges and stabilizes apposing membranes destined for fusion.
The McGill Inanimate System for Training and Evaluation of Laparoscopic Skills (MISTELS) was developed to assess laparoscopic skills and to score them objectively. This system has been described ...previously. The purpose of the current study was to determine a pass/fail threshold.
In this study, 165 individuals were tested and grouped according to their clinical competency in laparoscopic surgery. The noncompetent group consisted of medical students and surgical residents in their first 2 years of training (n = 83). The competent group consisted of chief general surgical residents in their last year of training, laparoscopy fellows, and practicing laparoscopic surgeons (n = 82). The Mann-Whitney U test was used to evaluate differences in task performance between the two groups.
There was a significant difference in total scores and individual MISTELS task scores between the noncompetent and competent laparoscopic surgeons (189 vs 372.5; p <0.0001). By setting specific pass/fail total score thresholds (cutoff scores), competent surgeons can be discriminated from noncompetent surgeons.
An objective pass/fail evaluation can be given to individuals tested with the MISTELS system.
PDF
