Biologically Inspired Engineering for Protein Stabilisation

Every year millions of lives are saved by vaccination and millions more could be saved with more efficient vaccination coverage. Thermostability of vaccines is one of the major reasons why many children who need vaccines most do not get all the shots they need. Vaccines need to be kept below 8°C throughout manufacturing, delivering and shipping which requires a cold-chain. This involves carrying vaccines in a cool box for days even weeks to small villages or islands with no electricity or any other infrastructure. One of the grand challenges in global health is to produce vaccines that do not require refrigeration. Here, a biologically inspired vaccine stabilisation method is proposed. The method is based on the fact that proteins entrapped in the fossil avian eggshell crystals are well preserved up to thousands of years in Africa where high-temperature vaccine stability is needed. The persistence of the ancient intra-crystalline proteins suggests that protein incorporation into the inorganic host could retard protein degradation for long periods of time without refrigeration. In the present work, biomimetic protein incorporation in calcium carbonate was investigated to produce intra-crystalline heat-stable vaccines. Intra-crystalline protein persistence in the fossil record has been shown for ostrich eggshell proteins and the most durable proteins are known to belong to the C-type lectin-like protein family. It is thought that at least one C-type lectin-like protein is found in the eggshells of every species. The most well known C-type lectin-like protein is the OC-17 from chicken eggshell. OC-17 and other C-type lectins have the highest concentration in eggshells compared to other eggshell proteins which suggests that they could lead understanding of efficient protein incorporation in calcium carbonate crystals. Apart from ostrich and chicken, C-type lectins from other species have not been studied in detail. For this reason, eggshells from 14 species were studied in the present work. The aim of the investigation is to quantify the effect of the organic matter on the eggshells which could allow inferring protein incorporation efficiency of different species. The mechanical properties of the eggshells were studied with nanoindentation. This allows probing the differences in the elastic modulus and hardness of eggshells which are affected by the intra-crystalline protein content. It was found that the elastic modulus differs among species, which is lowest at around 10 GPa for Bali myna and highest at around 60 GPa for ostrich. Similarly, the hardness changes from around 1 GPa for Bali myna to around 3 GPa for rhea. The chemical analysis was conducted with IR spectroscopy. The deviations in the absorption peaks of eggshells compared to pure calcium carbonate allows probing the extent of the amorphous structure of the eggshells. In addition, the comparison of the full-width at half maximum values of vibration modes in each spectrum provides information of the crystal order of the eggshells. Comparison of mechanical and chemical analyses of different species offers insights on the protein content of different eggshells which could lead identification of the most efficient C-type lectins in terms of protein incorporation ability. Because of the importance of eggshell C-type lectins for the vaccine preservation method proposed in the present work, the OC-17 was studied here in detail. First, a purification method was developed to extract OC-17 from chicken eggshell. Liquid chromatography was used to extract OC-17, OC-23 and lysozyme. Then, lysozyme was removed from solution using a lysozyme binding protein. In addition, OC-17 was cloned to synthesize a recombinant C-type lectin-like eggshell protein in bacteria for the first time. Growth conditions were optimized and OC-17 expression was verified with anti-Histag antibody. Lastly, the model protein incorporation into synthetic calcium carbonate was studied. Because the incorporated and reconstituted protein structure is of utmost importance, the secondary and tertiary protein structures of incorporated proteins were analyzed with circular dichroism and intrinsic tryptophan fluorescence spectroscopy. The effect of both the reconstitution and incorporation were studied independently as intra-crystalline proteins could denature during the interaction with the reconstitution buffer or during the incorporation process. It was shown that the model proteins BSA, lysozyme and diphtheria anti-toxin could be reconstituted using EDTA without structural change. The incorporation was found efficient for BSA only with an efficiency of %84 in terms of total amount of protein incorporated into the crystals. The secondary structure of BSA was shown to be stable during reconstitution and incorporation. A structural change in the tertiary structure was observed in BSA. Possible improvements to incorporate a target protein as a fusion construct using the OC-17 or using the ostrich intra-crystalline peptides as ’incorporation tags’ were discussed in order to move towards a real-world application of biomimetic vaccine stabilisation

Direct bioelectrocatalysis at the interfaces by genetically engineered dehydrogenase

This is the published version.There is an emerging interest in developing bio-functionalisation routes serving as platforms for assembling diverse enzymes onto material surfaces. Specifically, the fabrication of next-generation, laboratory-on-a-chip-based sensing and energy harvesting systems requires controlled orientation and organisation of the proteins at the inorganic interfaces. Herein, the authors take the initial steps towards designing multifunctional, enzyme-based platforms by genetically integrating the engineered materialselective peptide tags for tethering redox enzymes onto electrode surfaces. The authors engineered a fusion protein that genetically conjugates gold-binding peptide to formate dehydrogenase derived from Candida methylica. The expressed proteins were tested for both enzyme activity and self-directed gold-surface functionalisation ability. Their findings demonstrate the successful self-immobilisation of the engineered enzyme onto different gold electrodes while retaining the catalytic activity. Building on the functionalisation by the peptides, a fusion enzyme-integrated circuit-based biosensor system was designed. The catalytic conversion of the formate by the engineered dehydrogenase was successfully monitored on the electrode surface at subsequent intervals. The engineered peptide-mediated self-integrated electrode systems can be extended to develop a wide range of biosensing and energy-harvesting platforms using different combinations of materials and biomolecules. This paper contains supporting information that will be made available online once the issue is published. In the meantime, if you wish to get a copy of the supplementary file, please contact the Journals Editor, Sarah Brown, at [email protected]

Implications of intracrystalline OC17 on the protection of lattice incorporated proteins.

Acknowledgements: H. B. C. would like to thank the Cambridge Commonwealth, European & International Trust, the Department of Engineering, the Nanoscience Centre, and Prof Jim Huntington from the Cambridge Institute for Medical Research, Department of Haematology, Cambridge, UK.Biogenic CaCO3 formation is regulated by crystallization proteins during crystal growth. Interactions of proteins with nascent mineral surfaces trigger proteins to be incorporated into the crystal lattice. As a result of incorporation, these intracrystalline proteins are protected in the lattice, an example of which is ancient eggshell proteins that have persisted in CaCO3 for thousands of years even under harsh environmental conditions. OC17 is an eggshell protein known to interact with CaCO3 during eggshell formation during which OC17 becomes incorporated into the lattice. Understanding protein incorporation into CaCO3 could offer insights into protein stability inside crystals. Here, we study the protection of OC17 in the CaCO3 lattice. Using thermogravimetric analysis we show that the effect of temperature on intracrystalline proteins of eggshells is negligible below 250 °C. Next, we show that lattice incorporation protects the OC17 structure despite a heat-treatment step that is shown to denature the protein. Because incorporated proteins need to be released from crystals, we verify metal chelation as a safe crystal dissolution method to avoid protein denaturation during reconstitution. Finally, we optimize the recombinant expression of OC17 which could allow engineering OC17 for engineered intracrystalline entrapment studies

Exploring the Larvicidal and Repellent Potential of Taurus Cedar (<i>Cedrus libani</i>) Tar against the Brown Dog Tick (<i>Rhipicephalus sanguineus</i> sensu lato)

This study investigated the potential acaricidal and repellent effects of tar obtained from the Lebanon cedar (Cedrus libani A. Rich.) against the brown dog tick species Rhipicephalus sanguineus sensu lato Latreille (Acari: Ixodidae). The goal was to find an alternative, safe, and effective way to eliminate ticks. Tar is traditionally extracted from cedar trees in the Antalya region of Türkiye. The composition of the tar is primarily characterized by a diverse mixture of terpenes, with β-himachalene (29.16%), α-atlantone (28.7%), ar-turmerone (8.82%), longifolene-(V4) (6.66%), α-himachalene (5.28%), and β-turmerone (5.12%) emerging as the predominant constituents. The toxic effects of tar on tick larvae were studied through larval immersion tests (LIT), and its repellent activity was evaluated using a new larval repellent activity test (LRAT). The results revealed significant acaricidal effects, with mortality rates of 77.7% and 82.2% for the Konyaalti and Kepez strains of the brown dog tick, respectively, in response to a 1% concentration of tar. LC50 and LC90 values were determined as 0.47% and 1.52% for the Kepez strain and 0.58% and 1.63% for the Konyaalti strain, respectively. When comparing the repellent effect of tar to the widely used synthetic repellent DEET, repellency rates of up to 100% were observed. As a result, this study establishes, for the first time, the larvicidal and repellent effects of C. libani tar on ticks

Evaluation of 2015-2016 MOTAKK HBV DNA and HCV RNA External Quality Assessment National Program Results

MOTAKK, as a national external quality control program has been launched to evaluate the molecular detection of viral infections including HBV DNA and HCV RNA in molecular microbiology diagnostic laboratories in Turkey. This program is prepared in compliance with ISO 17043:2010 (Conformity assessment general requirements for proficiency testing) standards, and aims to take the place of external quality control programs from abroad, contributing to standardization and accuracy of molecular diagnostic tests in our country. The aim of this study was to evaluate 2015 and 2016 results of the MOTAKK External Quality Control Program for HBV DNA and HCV RNA viral load. The calls were announced on the web page of MOTAKK (www.motakk.org). The quality control samples were sent to participating laboratories in 2015 and 2016. Main stocks were prepared from patients with chronic hepatitis B and C who had viral load detection with reference methods according to WHO reference materials for viral load studies to improve quality control sera. From these main stocks, samples with different viral loads were prepared from dilutions of plasma with HBV, HCV, HAV, HIV, Parvovirus B19 and CMV negative serologic markers. Quality control samples were sent to the participating laboratories along with the negative samples in the cold chain. The laboratories accomplished the related tests within 2-3 weeks and entered their results on the MOTAKK web page. These results were analysed according to ISO 13528 (Statistical methods for use in proficiency testing by interlaboratory comparison) and scoring reports were created by a software developed by MOTAKK and sent to participating labs. Each laboratory evaluated their own results in comparison with the other laboratory results, reassessed the tests via observing the distance from the mean result and the reference values. The number of laboratories participating in the HBV DNA and HCV RNA external quality control program was 70-73 in 2015-2016. Participants were able to comply with the program tools, registering, entering results and receiving the results reports problem. In HBV panel, 72.6-89.1% and 84.7-90.3% of the participant laboratories were in 1 standard deviation (SD) in 2015-2016, respectively. In HCV panel, 70.8-89.1% and 84.7-90.3% of the participant laboratories were in 1 SD in 2015-2016, respectively. A national external quality control program for HBV DNA and HCV RNA in Turkey has been prepared for the first time with this project and implemented successfully. All the data provided in the MOTAKK external quality control program final report, compensate all the data provided by the quality control program final reports from abroad; additionally, the report allows comparison of used technologies and commercial products

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P &lt; 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)