Ion Mobility Spectrometry of Superheated Macromolecules at Electric Fields up to 500 Td.

Since its inception in 1980s, differential or field asymmetric waveform ion mobility spectrometry (FAIMS) has been implemented at or near ambient gas pressure. We recently developed FAIMS at 15-30 Torr with mass spectrometry and utilized it to analyze amino acids, isomeric peptides, and protein conformers. The separations broadly mirrored those at atmospheric pressure, save for larger proteins that (as predicted) exhibited dipole alignment at ambient but not low pressure. Here we reduce the pressure down to 4.7 Torr, allowing normalized electric fields up to 543 Td-double the maximum in prior FAIMS or IMS studies of polyatomic ions. Despite the collisional heating to ∼1000 °C at the waveform peaks, the proteins of size from ubiquitin to albumin survived intact. The dissociation of macromolecules in FAIMS appears governed by the average ion temperature over the waveform cycle, unlike the isomerization controlled by the peak temperature. The global separation trends in this "superhot" regime extend those at moderately low pressures, with distinct conformers and no alignment as theorized. Although the scaling of the compensation voltage with the field fell below cubic at lower fields, the resolving power increased and the resolution of different proteins or charge states substantially improved.

Candidate-gene association analysis for a continuous phenotype with a spike at zero using parent-offspring trios.

In this paper, we propose the class of generalized additive models for location, scale and shape in a test for the association of genetic markers with non-normally distributed phenotypes comprising a spike at zero. The resulting statistical test is a generalization of the quantitative transmission disequilibrium test with mating type indicator, which was originally designed for normally distributed quantitative traits and parent-offspring data. As a motivational example, we consider coronary artery calcification (CAC), which can accurately be identified by electron beam tomography. In the investigated regions, individuals will have a continuous measure of the extent of calcium found or they will be calcium-free. Hence, the resulting distribution is a mixed discrete-continuous distribution with spike at zero. We carry out parent-offspring simulations motivated by such CAC measurement values in a screening population to study statistical properties of the proposed test for genetic association. Furthermore, we apply the approach to data of the Genetic Analysis Workshop 16 that are based on real genotype and family data of the Framingham Heart Study, and test the association of selected genetic markers with simulated coronary artery calcification.

Healthy Dads, Healthy Kids UK, a weight management programme for fathers: feasibility RCT.

OBJECTIVE: To assess (1) the feasibility of delivering a culturally adapted weight management programme, Healthy Dads, Healthy Kids United Kingdom (HDHK-UK), for fathers with overweight or obesity and their primary school-aged children, and (2) the feasibility of conducting a definitive randomised controlled trial (RCT). DESIGN: A two-arm, randomised feasibility trial with a mixed-methods process evaluation. SETTING: Socioeconomically disadvantaged, ethnically diverse localities in West Midlands, UK. PARTICIPANTS: Fathers with overweight or obesity and their children aged 4-11 years. INTERVENTION: Participants were randomised in a 1:2 ratio to control (family voucher for a leisure centre) or intervention comprising 9 weekly healthy lifestyle group sessions. OUTCOMES: Feasibility of the intervention and RCT was assessed according to prespecified progression criteria: study recruitment, consent and follow-up, ability to deliver intervention, intervention fidelity, adherence and acceptability, weight loss, using questionnaires and measurements at baseline, 3 and 6 months, and through qualitative interviews. RESULTS: The study recruited 43 men, 48% of the target sample size; the mean body mass index was 30.2 kg/m2 (SD 5.1); 61% were from a minority ethnic group; and 54% were from communities in the most disadvantaged quintile for socioeconomic deprivation. Recruitment was challenging. Retention at follow-up of 3 and 6 months was 63%. Identifying delivery sites and appropriately skilled and trained programme facilitators proved difficult. Four programmes were delivered in leisure centres and community venues. Of the 29 intervention participants, 20 (69%) attended the intervention at least once, of whom 75% attended ≥5 sessions. Sessions were delivered with high fidelity. Participants rated sessions as 'good/very good' and reported lifestyle behavioural change. Weight loss at 6 months in the intervention group (n=17) was 2.9 kg (95% CI -5.1 to -0.6). CONCLUSIONS: The intervention was well received, but there were significant challenges in recruitment, programme delivery and follow-up. The HDHK-UK study was not considered feasible for progression to a full RCT based on prespecified stop-go criteria. TRIAL REGISTRATION NUMBER: ISRCTN16724454.

Ion Mobility Spectrometry of Macromolecules with Dipole Alignment Switchable by Varying the Gas Pressure.

Since inception in the 1980s, differential or field asymmetric waveform ion mobility spectrometry (FAIMS) was implemented at or near the ambient gas pressure (AP). Recently, we developed FAIMS at 15-30 Torr within a mass spectrometer and demonstrated it for small and medium sized ions, including peptides. The overall separation properties mirrored those at AP, reflecting the shared underlying physics. Here we extend these analyses to macromolecules, namely, multiply charged proteins generated by electrospray ionization. The spectra for smaller proteins (ubiquitin, cytochrome c, myoglobin) again resemble those at AP, producing features for one or a few adjacent well-defined conformers with type C behavior. Large proteins (single aldolase domain and albumin) now follow, with no broad bands for type A or B species that dominated at 1 atm. Those unique behaviors were ascribed to pendular ions with electric dipoles reversibly locked by the strong field in FAIMS. Disappearance of those bands shows loss of alignment predicted by first-principles theory, further supporting dipole locking at AP. The capability to modulate dipole alignment by varying gas pressure at constant normalized field provides the basis for determining the ion dipole moment and direction within the molecular frame from the pressure of onset and characteristics of spectral drift. This new approach to alter FAIMS separations of proteins could make a powerful tool for structural biology and be useful for proteomics and imaging.

Digital Affinity in Medical Students Influences Learning Outcome: A Cluster Analytical Design Comparing Vodcast With Traditional Lecture.

BACKGROUND/OBJECTIVE: Undergraduate medical education still relies on lectures as the core teaching activity. However, e-learning and new media have begun to augment learning and information gathering over the last few years. The aim of this study was to investigate the effect of 2 teaching formats in surgical education, a classic lecture and a video podcast (vodcast), on knowledge gain, in particular with respect to the participants' characteristics and preferences. DESIGN: A prospective study was conducted over 2 consecutive semesters. A traditional lecture on goitre was given to the first of the 2 semesters and replaced by a matching vodcast made available to the second. An untaught subject (cholelithiasis) served as control. Knowledge gain was calculated as the difference in point scores between entry and mid-module examinations. Furthermore, participants completed a postintervention survey, in which they specifically rated their digital affinity and learning preferences. A cluster analysis was conducted pooling both semesters to evaluate differences between individuals affecting their performance. RESULTS: Both teaching formats resulted in a significant knowledge gain. Two clusters could be identified across both semesters: Cluster 2 (Digital natives) proved to be significantly different from Cluster 1 (Traditional) with respect to the 4 variables: "technically interested," the "use of smartphones," "activity in social networks," and "reading in digital formats." The knowledge gain differences between formats for students in the "Traditional" cluster were statistically insignificant. However, students in the cluster "Digital natives" performed significantly worse when exposed to the lecture format. CONCLUSIONS: Cluster analysis revealed that the students with an obvious affinity to information communication technology were found to be at a significant disadvantage in the lecture. In future, we recommend offering some form of pretest to determine an individual's profile and empower students to plan their learning activities accordingly.

Differential Ion Mobility Separations in the Low-Pressure Regime.

Ion mobility spectrometry (IMS) in conjunction with mass spectrometry (MS) has emerged as a powerful platform for biological and environmental analyses. An inherent advantage of differential or field asymmetric waveform IMS (FAIMS) based on the derivative of mobility vs electric field over linear IMS based on absolute mobility is much greater orthogonality to MS. Effective coupling of linear IMS to MS and diverse IMS/MS arrangements and modalities impossible at ambient buffer gas pressure were enabled at much reduced pressures. In contrast, FAIMS devices operate at or near atmospheric pressure, which complicated integration with MS. Here, we show FAIMS at ∼15-30 Torr using a planar-gap stage within the MS instrument envelope. Fields up to ∼300 Td permitted by the Paschen law at these pressures greatly raise the separation speed, providing fair resolution in ∼10 ms and FAIMS scans in under 5 s. Rapid separation and efficient ion collection at low pressure minimize losses in the FAIMS step. Separations for key analyte classes and their dependences on electric field mirror those at ambient pressure. The potential for proteomics is demonstrated by separations of isomeric peptides with variant localization of post-translational modifications.

Reaching consensus on reporting patient and public involvement (PPI) in research: methods and lessons learned from the development of reporting guidelines.

INTRODUCTION: Patient and public involvement (PPI) is inconsistently reported in health and social care research. Improving the quality of how PPI is reported is critical in developing a higher quality evidence base to gain a better insight into the methods and impact of PPI. This paper describes the methods used to develop and gain consensus on guidelines for reporting PPI in research studies (updated version of the Guidance for Reporting Patient and Public Involvement (GRIPP2)). METHODS: There were three key stages in the development of GRIPP2: identification of key items for the guideline from systematic review evidence of the impact of PPI on health research and health services, a three-phase online Delphi survey with a diverse sample of experts in PPI to gain consensus on included items and a face-to-face consensus meeting to finalise and reach definitive agreement on GRIPP2. Challenges and lessons learnt during the development of the reporting guidelines are reported. DISCUSSION: The process of reaching consensus is vital within the development of guidelines and policy directions, although debate around how best to reach consensus is still needed. This paper discusses the critical stages of consensus development as applied to the development of consensus for GRIPP2 and discusses the benefits and challenges of consensus development.

Biographic Characteristics and Factors Perceived as Affecting Female and Male Careers in Academic Surgery: The Tenured Gender Battle to Make It to the Top.

PURPOSE: Given the high attrition rate in the field of academic surgery, we aimed to characterise the professional and personal situations of female and male academic surgeons as well as to gather data on their respective perceptions of career advancement and work satisfaction. METHODS: We conducted a cross-sectional survey in Germany, inviting all identifiable academically highly qualified female surgeons and their male counterparts in a 1:2 ratio to participate. An anonymous 103-item online questionnaire was designed and the data collected between July and September 2014. RESULTS: The questionnaire was sent to 93 female and 200 male surgeons, of whom 63 women (67.7%) and 70 men (35.0%) replied. The average age was 47.5 and 47.1 years, respectively. Respondents identified 'high degree of expertise', 'ambition', and 'clarity of one's professional aims' as important factors affecting professional career development. Both groups felt 'workload', 'working hours/shifts', and 'gender' to be a hindrance, the latter of significantly greater importance to female surgeons. The mean work satisfaction scores were high in both female (69.5%) and male (75.7%) surgeons. The predictors 'support from superiors' (standardised ß coefficient = 0.41) and 'manual aptitude' (ß = 0.41) contributed incrementally to the variance in 'high degree of work satisfaction' (90-100%) observed for female surgeons. However, childcare provided by 'kindergarten/crèche/after-school care' had the greatest negative predictive value (ß = -1.33). CONCLUSIONS: Although there are many parallels, female faculty members experience the culture of academic surgery to some extent differently from their male counterparts, especially when impacted by parenthood and childcare. Faculty development programmes need to develop strategies to improve perceived equality in career opportunities by respecting individuals' requirements as well as offering gender-appropriate career guidance.

Standardized and quality-assured video-recorded examination in undergraduate education: informed consent prior to surgery.

BACKGROUND: Communication skills combined with specialized knowledge are fundamental to the doctor-patient relationship in surgery. During a single-station video-recorded objective structured clinical examination (VOSCE), students were tasked with obtaining informed consent. Our aim was to develop a standardized and quality-assured assessment method in undergraduate education. METHODS: One hundred fifty-five students in their fifth year of medical school (78 videos) participated in a summative VOSCE within the framework of the teaching module "Operative Medicine." They prepared for three clinical scenarios and the surgical procedures involved. The examination comprised participants having to obtain informed consent from simulated patients, video recording their performance. Students were assessed by two independent raters, the background of one of whom was nonsurgical. Results were statistically tested using SPSS. RESULTS: Students' scores were all beyond the pass mark of 70%, averaging 91.0% (±4.0%), 88.4% (±4.4%), and 87.0% (±4.7%) for the appendectomy, cholecystectomy, and inguinal hernia repair checklist, respectively. Most items (68%-89% of the checklists) were found to have fair to excellent discrimination values. Cronbach's α values ranged between 0.565 and 0.605 for the individual checklists. Interrater agreement was strong (Pearson correlation coefficient = 0.80, P < 0.01; intraclass correlation coefficient 2.1 = 0.78). CONCLUSIONS: The VOSCE is both feasible and reliable as a method of assessing student communication skills and the application of clinical knowledge while obtaining informed consent in surgery. This method is efficient (flexible rating outside normal working hours possible with reductions in administrative load) and may be used for high-stakes evaluation of student performance.

Digital asymmetric waveform isolation (DAWI) in a digital linear ion trap.

Traditionally, in a quadrupole mass filter, ion isolation is achieved by scanning the rf and DC voltages with a fixed ratio. In this paper, we describe an innovative procedure implemented in a digitally driven linear ion trap termed digital asymmetric waveform isolation (DAWI) in which ion isolation is obtained by manipulation of the duty cycle of the rectangular waveforms. Variation of the waveform duty cycle allows introduction of a precisely defined DC quadrupole component into the main trapping field of the quadrupole ion filter. The DAWI method is completely controlled at software level and does not require any hardware modification.

Noninvasive imaging of liver repopulation following hepatocyte transplantation.

Near infrared fluorescence (NIRF) optical imaging is a technique particularly powerful when studying in vivo processes at the molecular level in preclinical animal models. We recently demonstrated liver irradiation under the additional stimulus of partial hepatectomy as being an effective primer in the rat liver repopulation model based on hepatocyte transplantation. The purpose of this study was to assess optical imaging and the feasibility of donor cell expansion tracking in vivo using a fluorescent probe. Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with irradiation. Four days later, a partial hepatectomy was performed and wild-type (DPPIV+) hepatocytes were transplanted into recipient livers via the spleen. Repopulation by transplanted DPPIV+ hepatocytes was detected in vivo with Cy5.5-conjugated DPPIV antibody using the eXplore Optix System (GE HealthCare). Results were compared with nontransplanted control animals and transplanted animals receiving nonspecific antibody. Optical imaging detected Cy5.5-specific fluorescence in the liver region of the transplanted animals, increasing in intensity with time, representing extensive host liver repopulation within 16 weeks following transplantation. A general pattern of donor cell multiplication emerged, with an initially accelerating growth curve and later plateau phase. In contrast, no specific fluorescence was detected in the control groups. Comparison with ex vivo immunofluorescence staining of liver sections confirmed the optical imaging results. Optical imaging constitutes a potent method of assessing the longitudinal kinetics of liver repopulation in the rat transplantation model. Our results provide a basis for the future development of clinical protocols for suitable fluorescent dyes and imaging technologies.

Digital ion trap mass spectrometer for probing the structure of biological macromolecules by gas phase X-ray scattering.

Small-angle X-ray scattering is a technique for the characterization and structural analysis of a variety of materials including biological macromolecules and polymers. For the conformational analysis of proteins, the interaction between sample and X-rays is generally performed when the proteins are present in solution. Here a three-dimensional digital ion trap interfaced with a high intensity X-ray source is built to prove that X-ray scattering can be performed on ions isolated in gas-phase. Initial experiments on an unresolved ion population of multiply charged cytochrome C ions indicate that a small-angle X-ray scattering signal can be detected and that partial structural information can be extracted about the overall molecular structure of protein ions.

Noninvasive Imaging of Liver Repopulation following Hepatocyte Transplantation.

Near infrared fluorescence (NIRF) optical imaging is a technique particularly powerful when studying in vivo processes at the molecular level in preclinical animal models. We recently demonstrated liver irradiation under the additional stimulus of partial hepatectomy as being an effective primer in the rat liver repopulation model based on hepatocyte transplantation. The purpose of this study was to assess optical imaging and the feasibility of donor cell expansion tracking in vivo using a fluorescent probe. Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with irradiation. Four days later, a partial hepatectomy was performed and wild-type (DPPIV+) hepatocytes were transplanted into recipient livers via the spleen. Repopulation by transplanted DPPIV+ hepatocytes was detected in vivo with Cy5.5-conjugated DPPIV antibody using the eXplore Optix™ System (GE HealthCare). Results were compared with nontransplanted control animals and transplanted animals receiving nonspecific antibody. Optical imaging detected Cy5.5-specific fluorescence in the liver region of the transplanted animals, increasing in intensity with time, representing extensive host liver repopulation within 16 weeks following transplantation. A general pattern of donor cell multiplication emerged, with an initially accelerating growth curve and later plateau phase. In contrast, no specific fluorescence was detected in the control groups. Comparison with ex vivo immunofluorescence staining of liver sections confirmed the optical imaging results. Optical imaging constitutes a potent method of assessing the longitudinal kinetics of liver repopulation in the rat transplantation model. Our results provide a basis for the future development of clinical protocols for suitable fluorescent dyes and imaging technologies.