2D-IR spectroscopy of proteins in H2O – a perspective

The form of the amide I infrared absorption band provides a sensitive probe of the secondary structure and dynamics of proteins in the solution phase. However, the frequency coincidence of the amide I band with the bending vibrational mode of H2O has necessitated the widespread use of deuterated solvents. Recently, it has been demonstrated that ultrafast 2D-IR spectroscopy allows the detection of the protein amide I band in H2O-based fluids, meaning that IR methods can now be applied to study proteins in physiologically relevant solvents. In this perspective, we describe the basis of the 2D-IR method for observing the protein amide I band in H2O and show how this development has the potential to impact on areas ranging from our fundamental appreciation of protein structural dynamics to new applications for 2D-IR spectroscopy in the analytical and biomedical sciences. In addition, we discuss how the spectral response of water, rather than being a hindrance, now provides a basis for new approaches to data pre-processing, standardisation of 2D-IR data collection and signal quantification. Ultimately, we visualise a direction of travel towards the creation of 2D-IR spectral libraries that can be linked to advanced computational methods for use in high-throughput protein screening and disease diagnosis

Members\u27 Percieved Benefits and Values of the Association for International Agricultural and Extension Education

The Association for International Agricultural and Extension Education (AIAEE) was established to strengthen and advance the knowledge base of agricultural and extension education worldwide. The purpose of this study was to determine perceived benefits of AIAEE membership. The objectives were to assess AIAEE members’ perceived organizational benefits; rank the top five perceived benefits; and, assign monetary values to the top five perceived benefits. AIAEE members (N= 161) responded to an online questionnaire. Respondents’ perceived organizational benefits included innovative ideas, professional knowledge, collaboration, opportunities to publish in the JIAEE (Journal of International Agricultural and Extension Education), and the importance of recognition awards. Their top five benefits were ranked as (a) journal, (b) conference, (c) networking, (d) professional development, and (e) communications. Lastly, they assigned monetary values to their AIAEE membership benefits. Values were highest for the journal (M = $25.06), followed by the conference (M =$21.94), and communications (M = $9.63). The AIAEE can recruit and retain members more effectively by continually evaluating members’ perceived benefits and values derived from membership. The AIAEE should create recruitment and retention plans based on members’ benefits and values, highlighting the most valuable and/or important benefits, and seek to strengthen lesser-valued benefits. As the AIAEE’s membership base changes, so too might change individual member’s perceived benefits and values, causing organizational shifts and/or priorities. For example, future AIAEE members may demand more social media interaction, instead of current communication mediums (i.e., journal, listserv, etc.

2D-infrared spectroscopy of proteins in water : using the solvent thermal response as an internal standard

Ultrafast two-dimensional infrared (2D-IR) spectra can now be obtained in a matter of seconds, opening up the possibility of high-throughput screening applications of relevance to the biomedical and pharmaceutical sectors. Determining quantitative information from 2D-IR spectra recorded on different samples and different instruments is however made difficult by variations in beam alignment, laser intensity, and sample conditions. Recently, we demonstrated that 2D-IR spectroscopy of the protein amide I band can be performed in aqueous (H2O) rather than deuterated (D2O) solvents, and we now report a method that uses the magnitude of the associated thermal response of H2O as an internal normalization standard for 2D-IR spectra. Using the water response, which is temporally separated from the protein signal, to normalize the spectra allows significant reduction of the impact of measurement-to-measurement fluctuations on the data. We demonstrate that this normalization method enables creation of calibration curves for measurement of absolute protein concentrations and facilitates reproducible difference spectroscopy methodologies. These advances make significant progress toward the robust data handling strategies that will be essential for the realization of automated spectral analysis tools for large scale 2D-IR screening studies of protein-containing solutions and biofluids

“Give us the tools!” - development of knowledge transfer tools to support the involvement of patient partners in the development of clinical trial protocols with patient-reported outcomes (PROs), in accordance with SPIRIT-PRO extension

Objectives (a) To adapt the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT)-patient-reported outcome (PRO) Extension guidance to a user-friendly format for patient partners and (b) to codesign a web-based tool to support the dissemination and uptake of the SPIRIT-PRO Extension by patient partners.Design A 1-day patient and public involvement session.Participants Seven patient partners.Methods A patient partner produced an initial lay summary of the SPIRIT-PRO guideline and a glossary. We held a 1-day PPI session in November 2019 at the University of Birmingham. Five patient partners discussed the draft lay summary, agreed on the final wording, codesigned and agreed the final content for both tools. Two additional patient partners were involved in writing the manuscript. The study compiled with INVOLVE guidelines and was reported according to the Guidance for Reporting Involvement of Patients and the Public 2 checklist.Results Two user-friendly tools were developed to help patients and members of the public be involved in the codesign of clinical trials collecting PROs. The first tool presents a lay version of the SPIRIT-PRO Extension guidance. The second depicts the most relevant points, identified by the patient partners, of the guidance through an interactive flow diagram.Conclusions These tools have the potential to support the involvement of patient partners in making informed contributions to the development of PRO aspects of clinical trial protocols, in accordance with the SPIRIT-PRO Extension guidelines. The involvement of patient partners ensured the tools focused on issues most relevant to them

Measuring proteins in H2O using 2D-IR spectroscopy; pre-processing steps and applications towards a protein library

The ability of two-dimensional infrared (2D-IR) spectroscopy to measure the amide I band of proteins in H2O rather than D2O-based solvents by evading the interfering water signals has enabled in-vivo studies of proteins under physiological conditions and in biofluids. Future exploitation of 2D-IR in analytical settings, from diagnostics to protein screening, will however require comparisons between multiple datasets, necessitating control of data collection protocols to minimise measurement-to-measurement inconsistencies. Inspired by analytical spectroscopy applications in other disciplines, we describe a workflow for pre-processing 2D-IR data that aims to simplify spectral cross-comparisons. Our approach exploits the thermal water signal that is collected simultaneously with, but is temporally separated from the amide I response to guide custom baseline correction and spectral normalisation strategies before combining them with Principal Component noise reduction tools. Case studies show that application of elements of the pre-processing workflow to previously-published data enables improvements in quantification accuracy and detection limits. We subsequently apply the complete workflow in a new pilot study, testing the ability of a prototype library of 2D-IR spectra to quantify the four major protein constituents of blood serum in a single, label-free measurement.  These advances show progress towards the robust data handling strategies that will be necessary for future applications of 2D-IR for pharmaceutical or biomedical applications

Citizen science to improve patient and public involvement in GUideline Implementation in oral health and DEntistry (the GUIDE platform)

Background: Citizen science is a way to democratise science by involving groups of citizens in the research process. Clinical guidelines are used to improve practice, but their implementation can be limited. Involving patients and the public can enhance guideline implementation, but there is uncertainty about the best approaches to achieve this. Citizen science is a potential way to involve patients and the public in improving clinical guideline implementation. We aimed to explore the application of citizen science methods to involve patients and the public in the dissemination and implementation of clinical guidelines in oral health and dentistry. Methods: We developed GUIDE (GUideline Implementation in oral health and DEntistry), a citizen science online platform, using a participatory approach with researchers, oral health professionals, guideline developers and citizens. Recruitment was conducted exclusively online. The platform focused on prespecified challenges related to oral health assessment guidelines, and asked citizens to generate ideas, as well as vote and comment on other citizens' ideas to improve those challenges. Citizens also shared their views via surveys and two online synchronous group meetings. Data were collected on participant's demographics, platform engagement and experience of taking part. The most promising idea category was identified by an advisory group based on engagement, feasibility and relevance. We presented quantitative data using descriptive statistics and analysed qualitative data using inductive and deductive thematic analysis. Results: The platform was open for 6 months and we recruited 189 citizens, from which over 90 citizens actively engaged with the platform. Most citizens were over 34 years (64%), female (58%) and had a university degree (50%). They generated 128 ideas, 146 comments and 248 votes. The challenge that led to most engagement was related to prevention and oral health self-care. To take this challenge forward, citizens generated a further 36 ideas to improve a pre-existing National Health Service oral care prevention leaflet. Citizens discussed motivations to take part in the platform (understanding, values, self-care), reasons to stay engaged (communication and feedback, outputs and impact, and relevance of topics discussed) and suggestions to improve future platforms. Conclusion: Citizen science is an effective approach to generate and prioritise ideas from a group of citizens to improve oral health and dental services. Prevention and oral health self-care were of particular interest to citizens. More research is needed to ensure recruitment of a diverse group of citizens and to improve retention in citizen science projects. Patient or Public Contribution: This project was inherently conducted with the input of public partners (citizen scientists) in all key aspects of its conduct and interpretation. In addition, two public partners were part of the research team and contributed to the design of the project, as well as key decisions related to its conduct, analysis, interpretation and dissemination and are co-authors of this manuscript.</p

Bioprocess monitoring applications of an innovative ATR-FTIR spectroscopy platform

Pharmaceutical manufacturing is reliant upon bioprocessing approaches to generate the range of therapeutic products that are available today. The high cost of production, susceptibility to process failure, and requirement to achieve consistent, high-quality product means that process monitoring is paramount during manufacturing. Process analytic technologies (PAT) are key to ensuring high quality product is produced at all stages of development. Spectroscopy-based technologies are well suited as PAT approaches as they are non-destructive and require minimum sample preparation. This study explored the use of a novel attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy platform, which utilises disposable internal reflection elements (IREs), as a method of upstream bioprocess monitoring. The platform was used to characterise organism health and to quantify cellular metabolites in growth media using quantification models to predict glucose and lactic acid levels both singularly and combined. Separation of the healthy and nutrient deficient cells within PC space was clearly apparent, indicating this technique could be used to characterise these classes. For the metabolite quantification, the binary models yielded R2 values of 0.969 for glucose, 0.976 for lactic acid. When quantifying the metabolites in tandem using a multi-output partial least squares model, the corresponding R2 value was 0.980. This initial study highlights the suitability of the platform for bioprocess monitoring and paves the way for future in-line developments

Sensing of bacterial spores with 2D-IR spectroscopy

Ultrafast 2D-IR spectroscopy has proved to be a powerful analytical tool for the detection and differentiation of Bacillus spores as dry films on surfaces. Here, we expand on these findings by employing 2D-IR spectroscopy to study spores from B. atrophaeus (BG) in aqueous solution. Specific vibrational modes attributable to the calcium dipicolinate trihydrate biomarker for spore formation were observed alongside distinctive off-diagonal spectral features that can be used to differentiate spores from different Bacillus species, indicating that 2D-IR has potential for use as a sensing platform with both solid and liquid phase samples. The ability of 2D-IR to enhance the protein amide I band relative to the overlapping water bending vibration was exploited to compare the nature of the protein component of spores to that of solution phase protein molecules. The vibrational lifetime for the amide I band of the BG spore in H2O was 1.4 ± 0.1 ps, longer than those reported for the proteins in H2O solution. The nature of a band at 1710 cm-1 was also investigated. Collectively these results show the potential advantages of 2D-IR spectroscopy, with successful detection and classification of spores under different conditions being based on detailed molecular understanding of the spore state