A historical and proteomic analysis of botulinum neurotoxin type/G

<p>Abstract</p> <p>Background</p> <p><it>Clostridium botulinum </it>is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an <it>in silico </it>and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity <it>in vitro</it>. In addition, we review previous literature to provide a complete description of the BoNT/G complex.</p> <p>Results</p> <p>An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs--nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison.</p> <p>Conclusions</p> <p>The <it>in silico </it>protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of <it>C. botulinum</it>. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MS^Edata analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.</p

Mass Spectrometric Analysis of Multiple Pertussis Toxins and Toxoids

Bordetella pertussis (Bp) is the causative agent of pertussis, a vaccine preventable disease occurring primarily in children. In recent years, there has been increased reporting of pertussis. Current pertussis vaccines are acellular and consist of Bp proteins including the major virulence factor pertussis toxin (Ptx), a 5-subunit exotoxin. Variation in Ptx subunit amino acid (AA) sequence could possibly affect the immune response. A blind comparative mass spectrometric (MS) analysis of commercially available Ptx as well as the chemically modified toxoid (Ptxd) from licensed vaccines was performed to assess peptide sequence and AA coverage variability as well as relative amounts of Ptx subunits. Qualitatively, there are similarities among the various sources based on AA percent coverages and MS/MS fragmentation profiles. Additionally, based on a label-free mass spectrometry-based quantification method there is differential relative abundance of the subunits among the sources

Botulinum Neurotoxin Detection and Differentiation by Mass Spectrometry

A new rapid, mass spectrometry-based method to detect and differentiate botulinal neurotoxins is described

Presenting a complex project assignment through selected video formats

The qualities and availability of different video formats offer many opportunities within the context of Higher Education (Hansch et al., 2015; Johnson et al., 2016; van Huystee, 2016). There is a shift within Higher Education to transition from the traditional face to face approach, to a more ‘blended’ approach in which face to face and online delivery of content are blended (Bates, 2015). More delivery of content is now provided online in video format, viewed before the class, as part of a flipped classroom (Bishop & Verleger, 2013; Yousef, Chatti, & Schroeder, 2014) and this is impacting the traditional role of the lecturer from ‘sage on the stage’, to ‘guide on the side’ (Tapscott, 2009). When creating video, a lecturer needs to have an understanding of the particular pedagogic affordances of the different types of video (Koumi, 2014; Thomson, Bridgstock, & Willems, 2014) and to know how to implement and embed these effectively into the teaching environment as part of a blended approach (Dankbaar, Haring, Moes, & van Hees, 2016; Fransen, 2006; Woolfitt, 2015). There needs to be awareness of how to embed the video from a didactic perspective to create meaningful learning (Karppinen, 2005) and an understanding of some of the financial and technical issues which include the relationship between cost of video production and the user experience (Hansch et al., 2015) and creating the correct combination of multimedia visual and audio elements (Colvin Clark & Mayer, 2011). As the role of the lecturer changes, there are a number of challenges when navigating through this changing educational environment. Massive Open Online Courses (MOOCs) provide lots of data for analysis and research shows that students in this environment stop watching videos after about six minutes (Guo, Kim, & Rubin, 2014) and that the most common video style used in MOOCs was the talking head with Power Point (Reutemann, 2016). Further research needs to be conducted regarding student preferences of video styles and correlation between video styles and course drop-out rates. As part of its research, the Inholland research group ‘Teaching, Learning and Technology’ (TLT) examines the use of ICT and video to support teaching and learning within Inholland. In 2015-2016, several pioneers (Fransen, 2013) working at Inholland explored different approaches to using video to support the teaching and learning process within a number of educational environments. TLT supported the pioneers in establishing their role within their faculty, creating a framework within which the pioneer can design the video intervention, collecting data and reflecting on what was learned through this process. With some of the projects, a more formal research process was followed and a full research report could be compiled. In other cases, the pioneer took a more exploratory and experimental approach. In these cases, the pioneer may not have conducted the video intervention under a formal research framework. However, during this process the pioneer may have uncovered interesting and valuable practical examples that can inspire and be shared with other educators. This current report falls under the category Research Type 3 as defined by TLT. It describes and assesses an ICT application (in this case, video) in order to share the original approach that could have high potential to be implemented in a broader educational context

Live Webinar on video teaching

On February 23, 2016, Zac Woolfitt gave a live webinar on video teaching. He was located at Inholland Diemen (where he also teaches Tourism), the broadcast was directly transferred to the Sonic Foundry worldwide users group, based in Madison, Wisconsin, USA

Using video to support teaching and learning on the ‘Levensbeschouwing’ minor: Research report

Video was used in a variety of formats during a third year elective Inholland course ‘Levensbeschouwing’ at the Faculty Onderwijs en Innovatie (education and innovation) in Amstelveen. From April to June (2017), 30 part-time and full-time students on the Tweedegraads Leraren Opleiding chose to follow the course for the ten week study period. Students used a variety of video formats to support and enhance their learning process. This research evaluates how the didactic embedding of video supported the teaching and learning on the course. The objective of the research was to gain insight into the ways in which video supports the learning and teaching process in the course and into the perceived practicality and effectiveness of this video support. The research examined how video was integrated into the course structure, how students and lecturers perceived the practicality of the use of video, and whether they considered it effective. The conversational framework of Laurillard (2002), was used as a framework in which to examine the interaction between students and teachers at the conceptual and application level. An overview was made of the different forms of videos used during the course (both teacher and student generated), how they were viewed and the value of them to the students. A survey was collected on the last day of the course in which students could share feedback on the ways in which video had contributed to their learning process. At the end of the course, a group interview was held with six student representatives (one from each group) and with the lecturers on the course to collect additional qualitative feedback on how video contributed to the learning process

Verrijking van de leeromgeving door de inzet van video: Animatie

Video kan in allerlei vormen de leeromgeving verrijken en daarmee van toegevoegde waarde zijn in een leerproces. Het gebruik van video heeft voordelen voor de student en voor jou als docent. Het lectoraat Teaching, Learning & Technology van hogeschool Inholland doet onderzoek naar de inzet van video bij leerprocessen. Deze animatie gaat in op wat je moet doen om de verschillende typen video te kunnen gebruiken