Subsequent Use Research Articles

Fungal bioprocessing of wheat straw with fruit and vegetable discards to produce cattle feeds for enhanced sustainability

Feed production for livestock requires abundant resources which compete with human food production. However, the food supply chain unintendedly generates a large amount of food loss and waste that impedes sustainability. This study tested the utility of making cattle feeds from wheat straw (WS) and fresh fruit and vegetable (FFV) discards through fungal bioprocessing. Two food-grade fungi, Tramates versicolor and Pleurotus ostreatus demonstrated high efficacy in degrading lignin (34–48 % and 16–29 % reduction, respectively) in fermented WS. Co-fermenting WS with FFV resulted in a large reduction of soluble nutrients from FFV, which were assimilated into fungal biomass for subsequent use in animal feed. Changes in the nitrogen profile occurred, with a decrease in nitrate and soluble proteins and increases in essential amino acids. These results demonstrate the potential for improving energy and protein value of crop residues and fruit/vegetable discards via fungal fermentation to achieve more circular food production and a more sustainable food future.

Exploring the intersection of sexual identity and route of administration in relation to cannabis use among young adult females.

Rates of cannabis use continue to increase with sexual minority women (SMW) reporting greater use than heterosexual women. Along with these increasing trends, the routes of administration (ROA) for cannabis are evolving. The current study examined associations between cannabis ROA and frequency of use, as well as differences across sexual identity (heterosexual vs. SMW). Participants were 949 young adult (18-25 years old) women (29.8% SMW) who reported past month cannabis use and were recruited through Amazon Mechanical Turk. Number of cannabis use days and each ROA used (joint, pipe, blunt, bong, vape, edible, and ointment) in the past 30 days were measured. Analysis of covariance models examined if sexual identity moderated the association between each ROA and cannabis use frequency. Among the full sample, joints were the most common ROA (78.6%); cannabis vaping was the most common noncombustible ROA (25.9%). SMW were more likely than heterosexual women to use each ROA except for joints. SMW who used pipes or edibles reported greater cannabis use frequency, compared to those who did not; there were no differences in frequency of use across ROA for heterosexual women. SMW may use a greater variety of ROA, potentially increasing the harms associated with cannabis. Marketing strategies targeting the sexual minority community may increase the likelihood of using various cannabis ROA and subsequent use. Findings further our knowledge about how young adult women are using cannabis, and highlight how ROA may contribute to the disparities observed among SMW.

Simultaneous removal of phosphorus and organic matter in municipal tail water enhanced by Fe2+/S2O82- system: Parameter optimization, efficiency, mechanism

The simultaneous removal of phosphorus (TP) and organic matter is a very ingenious process and has attracted much attention. A Fe2+/S2O82- system was constructed to advanced treat TP and organic matter in sewage. The influencing factors and degradation mechanism were investigated. The results showed that the parameters of Fe2+/P and S2O82-/Fe2+ were optimized to be 5 and 4, respectively. The molar ratio of S2O82-/Fe2+ was 4, the removal performance of TP and target oriented curriculum (TOC) was the best, and the increase of temperature was beneficial to the removal of TP and TOC. The precipitate in the secondary effluent was mainly ferric phosphate. The TP removal mechanism lied in the removal by the formation of Fe4(PO4)3(OH)3 precipitation. And protein macromolecular organic matter is degraded into small molecular organic matter such as methane (CH4) for subsequent use as a high-quality carbon source.

Effect of aging on mechanical properties of 3D printed samples using stereolitography

This paper focuses on stereolithography (an additive manufacturing technology working on the principle of curing liquid resins layer by layer using ultraviolet radiation) and the effect of aging on the mechanical properties of the material and printed samples. The aging of the material could be a problem for its subsequent use as the stability of the mechanical properties would not be maintained and unwanted deterioration of the material could occur. As part of the research, sets of samples were printed and subjected to different aging methods and subsequently subjected to quasi-static and dynamic uni-axial load tests. From the data obtained, the basic mechanical properties of the material were calculated and compared with each other. The aim of this paper was to investigate whether aging process causes significant changes in the mechanical properties of the materials used, which could have a consequential impact on their use in different industries.

Effects of a nicotine warning label and vaping cessation resources on young adults’ perceptions of pro-vaping instagram influencer posts

BackgroundExposure to social media content promoting e-cigarette use (“vaping”) is associated with subsequent tobacco use among young adults. Adding features to pro-vaping Instagram influencer posts, such as a nicotine warning label and vaping cessation resources, could help counteract posts’ negative influence. MethodsYoung adults (N = 2,179; Mage = 22.6 [SD = 0.4]; 53.0 % cisgender women, 45.1 % Hispanic) completed an online experiment in 2021–2022 through an ongoing prospective cohort study. Participants viewed three simulated pro-vaping Instagram influencer posts in a four-group, between-subjects design. Post features differed by experimental condition: “label-only” (nicotine warning label on post), “link-only” (link to vaping cessation resources under post), “L&L” (label and link), or “control” (neither). Participants rated each influencer’s traits (honest, trustworthy, informed, smart, attractive, popular; 0–100 %). After viewing all three posts, participants reported use intentions, susceptibility, positive and negative expectancies, and harm perceptions around the fictitious advertised vaping product. Past-month vapers additionally reported their desire and self-efficacy for quitting. ResultsL&L (versus control and link-only) participants viewed influencers as more honest, trustworthy, and informed. L&L (versus control) participants had lower odds of susceptibility to using the advertised product, lower positive expectancies, and greater negative expectancies. The label and link did not significantly affect participants’ intentions to use the product, perceived harm of the product, or desire or self-efficacy for quitting vaping. ConclusionsProviding a nicotine warning label and link to vaping cessation resources on influencers’ Instagram posts may have the unintended effect of increasing positive perceptions of the influencer. However, they may reduce susceptibility to product use.

Holocene landscape reconstruction in the surroundings of the Temple of Pepi I at ancient Bubastis, southeastern Nile Delta (Egypt)

AbstractIn ancient Egypt, lakes, canals, and other water bodies were an essential part of the sacred landscape in which temples were embedded. In recent years, geoarchaeological research at the site of the Temple of Bastet at Bubastis in the southeastern Nile Delta has proven the existence of two water canals surrounding the temple. It has now been investigated whether these canals were connected to the Temple of Pepi I (2300–2250 B.C.E.), located approximately 100 m to the west of the Temple of Bastet. To explore the Holocene landscape genesis of the Temple of Pepi I, 15 drillings and six geoelectrical profile lines were performed in the surroundings of the temple in spring 2022. The results show loamy to clayey sediments in deeper sections of all drillings with a maximum thickness of 1.70 m, indicating a marshy or swampy depositional environment. Based on the recovered sediment sequences and archaeological remains in the vicinity of the Temple of Pepi I, the marshy or swampy area existed before the Fourth Dynasty. During the Old Kingdom (ca. 2850–2180 B.C.E.), the former marshland either dried up through natural processes or was intentionally drained and filled with sediments for subsequent use for occupation. Regarding the original research question, there is as yet no evidence for a direct connection to the canals of the Temple of Bastet.

Electricity produced using biogas obtained from the recovery of organic waste in ecological landfills – a case study

The paper presents an applied technological study regarding the production of biogas and its subsequent use for the production of electricity, at the level of a developed rural agro-zootechnical area.

Potential of Porous Substrate Bioreactors for Removal of Pollutants from Wastewater Using Microalgae.

Porous substrate bioreactors (PSBRs) are a new technology to grow microalgae immobilized in a dense culture and solve some problems linked to suspended cultivation. During recent years, this technology has been used in laboratory and pilot setups in different fields of environmental biotechnology, such as wastewater treatment. The aim of this short review is to introduce the PSBR technology, summarize the results obtained in removing some pollutants from wastewater, provide an assessment of the potential of PSBRs for wastewater treatment, and the subsequent use of the algal biomass for other purposes.

Meaning and Replicability of Social Psychological Experiments: A Personal Account

In this paper I describe the gradual evolution of experimental social psychology from a so called “science of discovery”, with invariant findings over time and space, to an art of theatrical reflection with self-made reproductions of social life to so engage in more thoughtful action. I do this by means of a narrative account of my sixty year long personal experience with the various changes in the discipline, from the many cases of non-replicability early in my career, to the splendid discovery by vigilant students at my department of the probably most impressive case of fraud in the history of the social sciences, to the persistent denial of a need for a new logic by orthodox positivists in the discipline, even after more than half of the carefully designed replications of important experiments in the discipline failed. I look at this evolution through the lens of gradual objectification in science as a whole, from the most distant objects, such as stars, to the closer and closer reality of our own existence, namely the production and processing of meaning, and the inevitable recursivity (historicity) and incompleteness (one cannot include one’s own discourse in the discourse one has at that moment) of that endeavour. I also describe moments of institutional evolution, its political besides scientific interest, and encounters with the most influential protagonists in the development of the new logic. I also reflect a moment on the theoretical and methodological implications of that new logic, and end with showing how the theme of social engagement and the move toward cultivated action, rather than by freezing history in the so called truth of invariance, was actually the core idea in the work of Kurt Lewin, and has been used inadvertently in the subsequent use of experiments in the discipline, even by those who pretended to be the astronomers of the mind. But with the clear note that reflection on social life in the form of self-made reproductions of that life, is probably the most human thing we can do, and enables us to create culture, rather than blind evolution in merely physical or biological terms. So, making experimental social psychology a valuable tool of humanization as well, but only when properly used.

Social Navigation for Adolescent Emergency Department Patients: A Randomized Clinical Trial

PurposeAdolescent emergency department (ED) patients have unmet social needs that contribute to ED use. This study aimed to evaluate the effect of social needs navigation for adolescents on subsequent ED visits and community resource use and to identify characteristics associated with elevated social risk. MethodsBetween July 2017 and August 2019, we used a random date generator to establish intervention and control group enrollment dates. All adolescents completed a social needs survey. Adolescents enrolled on intervention dates received in-person, risk-tailored social needs navigation. Those enrolled on control dates received a preprinted resource guide. We used chart review and follow-up calls to assess 12-month ED revisits and community resource use. Logistic regression was used to compare these outcomes between groups. We measured the association between ≥3 reported unmet needs and characteristics hypothesized a priori to be associated with elevated social risk (nonurgent visits, obesity, or any of nine “socially sensitive” chief complaints) using logistic regression. ResultsA total of 399 adolescents were randomized. There was no difference between groups in the number of ED revisits. There was increased community resource use in the intervention group (adjusted odds ratio [aOR]: 3.5 [95% confidence interval {CI}: 1.5, 8.2]). Adolescents with a socially sensitive chief complaint had increased odds of ≥3 unmet needs (aOR: 2.2 [95% CI: 1.3, 3.6]), as did those with food insecurity in a post hoc analysis (aOR: 9.9 [95% CI: 4.0, 24.6]). DiscussionSocial needs navigation increased community resource use but not subsequent ED visits. Adolescents with socially sensitive chief complaints or food insecurity reported increased unmet needs.

The intraoperative visualisation technique during lead implantation into the cardiac conductive system: aspects of computed tomography: prospective study

INTRODUCTION: The lead implantation into the cardiac conduction system (CCS) is the most physiological method of pacing nowadays. «The method of intraoperative visualization and control of the lead position for permanent electrocardiostimulation during implantation of the lead in the CCS» has been developed for reduce the number of non-targeted implantations. This method based on the integration into the angiograph system 3D-reconstruction of the heart converted to computed tomography (CT) in the form of a mask against the background of fluoroscopy. CT is an important stage of the intraoperative visualization technique (IVT).OBJECTIVE: The aim of the study was to adapt the protocol of CT examination of the heart with contrast to construct a partially segmented 3D-reconstruction of the heart on an angiographic complex for subsequent use during of the lead implantation in the CCS within the framework of the author’s IVT.MATERIALS AND METHODS: As part of the development of the IVT, 21 CT studies of the heart were selected from own database. The step of the gradient of the density difference of the contrasted blood is about 10 HU, the range of the difference of densitometric parameters of the «left ventricle (LV) — right ventricle (RV)» from 0 HU to 200 HU. As well as selected 11 CT studies of the heart. The step of the gradient of the difference of densitometric indicators the contrasted blood in «the RV cavity — myocardium» is about 10 HU, the range is from 0 HU to 100 HU. All CT scans are alternately loaded into the angiograph, followed by the creation of a 3D model of the heart using basic software.RESULTS: It’s necessary to exceed the degree of contrast of the LV cavity over the RV cavity by at least 80 HU to perform partial segmentation on the left and right chambers of a 3D-model of the heart in an angiographic complex that does not have a specialized segmentation module. A sufficiently large part of the left ventricular cavity (LV) disappears with a smaller gradient when the right ventricular cavity (RV) is suppressed. The minimum gradient of «the ventricular cavity — myocardium» is at least 20 HU. The boundaries of the right ventricular edge of the interventricular septum (IVS) are not visualized with a smaller contrast gradient. It’s important for determining the insertion place of the lead into the IVS.CONCLUSION: It’s necessary to exceed the contrast of the LV cavities above the RV cavity by at least 80 HU, the RV cavity above the myocardium by at least 20 HU to perform partial segmentation on the left and right chambers of a 3D-model of the heart in an angiographic complex that does not have a specialized segmentation module

Study of the process of obtaining water glass from silica gel for use in metallurgy

This study explores the possibility of producing a sodium silicate solution (liquid glass) using industrial raw materials for subsequent use in metallurgical practice. The object of the study was industrial waste from the production of aluminium fluoride, i.e., silica gel, which comprises a fine powder of silicon dioxide with a moisture content of over 55 wt %. Silica gel was purified using a low-concentration solution of sulfuric acid. The synthesis of liquid glass was carried out using the HEL Auto-Mate Reactor System. The silicon content in the solution was determined by X-ray fluorescence method using the Shimadzu EDX-7000P analyzer. To determine the alkali content in the resulting product, a titrimetric analysis method was used. The preliminary purification process allowed silica gel with an amorphous silica content of over 98 wt % to be obtained. The optimal parameters of the liquid glass production were defined: temperature – 100°C, process time – 4.5 hours, mixing speed – 300 rpm, and the concentration of the initial alkaline solution – from 10 to 17.5 wt %. The resulting liquid glass solution had a mass content of silicon dioxide from 16.65 to 23.77 wt % and a silicate module from 2.72 to 3.16, which meets the requirements of marketable products for various industries. Based on the experimental results, optimal parameters for the production of liquid glass using industrial raw materials, i.e., silica gel, are proposed. Liquid glass with the defined characteristics can be further used as a binder in metallurgical processes.

Hydroformylation of olefins over a novel active rhodium catalyst supported on a melamine–cyanuric acid co-crystal

Hydroformylation, or oxo synthesis, is one of the most important industrial petrochemical processes involving homogeneous catalysts. The development of efficient and stable phosphorus-free heterogeneous catalytic systems for this process is of great interest because it can solve the problem of separation and recycling of expensive metals and reduce the amount of phosphorus-containing waste. Macromolecular compounds containing triazine rings can be promising candidates for supports for the stabilization of metal nanoparticles and subsequent use in heterogeneous catalytic reactions. Herein, it was shown that the interaction of cyanuric chloride with melamine produces cyanuric acid–melamine (CAM) co-crystal, which was used as a support for stabilizing rhodium nanoparticles. The obtained catalyst (CAM/Rh) exhibits high activity and stability in the hydroformylation of olefins of various structures. After completion of the reaction, the catalyst is easily separated from the reaction mixture and can be used repeatedly with virtually no loss of activity.

Analytical research of the parameters and characteristics of new “quarry cavities – backfill material” systems: Case study of Ukraine

Purpose. The research is aimed to identify, investigate and study the parameters and characteristics of the “quarry cavities – backfill material” systems on the territory of Ukraine using new comprehensive methodological tools that form the basis for the development of effective methods for restoring the earth’s surface with an emphasis on industrial and construction use. Methods. An integrated approach is used, which includes: analysis of the spatial location of industrial waste heaps on the territory of Ukraine as potential backfill materials and resulting quarry cavities that are not subject to complete earth’s surface restoration, as well as determination of the volumes of resulting cavities and backfill materials. Based on a set of factors, the “quarry cavities – backfill material” systems have been ranked according to priority. Tools used are: information data from the State Informational Geological Fund of Ukraine, registers of industrial waste accumulation sites in region, the Google Earth satellite program, an online topographic map (OpenStreetMap) and Blender program for constructing 3D models. Findings. A new concept and theoretical representation of “quarry cavities – backfill material” has been formulated. The characteristics of a number of important conditions for the harmonious existence and effective implementation of promising “quarry cavities – backfill material” systems are provided. A new information-analytical map of the spatial location of potential backfill materials and resulting quarry cavities has been created. Thirteen promising systems and their parameters have been identified, within which it is appropriate to consider backfill technologies for the complete earth’s surface restoration. The existing balance of cavities and backfill materials for the identified systems has been determined, followed by grading of quarries according to the predicted lifespan. Originality. The “quarry cavities – backfill material” systems, which have the greatest advantages, are specified by ranking them according to a complex of technological, environmental, economic and social factors. Practical implications. The results obtained provide valuable information for the development of a government strategy and environmental programs for the restoration of land areas disturbed by mining operations based on backfill technologies and their subsequent use for industrial purposes.

In English

This article analyses some literature data and the authors’ developments in the technology of creating of therapeutic depots in the form of films, dispersions of metal oxides and textiles with immobilized biocompatible silver nanoparticles (NPs) in the structure of SiO2, TiO2, cotton, biopolymers (alginate, chitosan, lignin, etc.), that have biocidal action, and future trends in this area. We and other researchers have developed methods for the synthesis of photocatalytically active TiO2 and SiO2 films, modified with gold/silver/copper NPs, suitable for medical use. An economical and simple low-temperature methods of manufacturing antimicrobial textiles by photo- or thermal activation and the possibility of their multiple use have been developed. The production of biomedical textiles is recently focused on the widespread use of non-toxic biopolymers, combined with textile. We have obtained compositions based on nanodispersed silica with polysaccharide sodium alginate and silver NPs with pronounced hemostatic and bactericidal properties. Obtaining a hybrid material based on a bactericidal textile combined with a dispersed oxide is promising for additional absorption of toxins and wound cleaning. The creation of such universal multifunctional materials includes their high bactericidal and antiviral multiply use. Hybrid materials based on metal NPs in the structure of carriers of different nature as films and dispersions of biocompatible oxides, biopolymers, textiles have a protection against possible toxic effects of nanoparticles and metal ions, self-cleaning capability, photocatalytic, hemostatic properties, temperature resistance, and other. The development and application of such materials is growing rapidly. So, materials based on Ag/SiO2 dispersions have high antibacterial and antiviral action (single application). Ag/SiO2 films can act as durable antibacterial cover. There is an enhancement in the antibacterial properties of Ag-TiO2 NPs under visible light irradiation and the photocatalytic effect under UV light (single application in the powder form). Self-cleaning, antimicrobial and UV-protective properties have Ag-TiO2 NPs in textile. Cotton modified with MeNPs demonstrates high efficiency of destruction of bacteria E. coli, K. pneumoniae, E. aerogenes, P. vulgaris, S. aureus, C. albicans, etc., with saving of biocidal activity after 5 cycles of washing. The dynamics of silver ions release from the surface of NPs in the structure of textile upon their contact with water for 72 hours have been studied. The number of irreversibly bound particles in textile structure is sufficient for subsequent use. Modified fabrics are reusable. Composites based on metal NPs in the structure of silica or titania in the presence of biopolymers are effective hemostatic agents with a bactericidal effect. Sodium alginate has a reducing and stabilizing effect on nanoparticles, and silica prevents agglomeration of metal NPs in the resulting composite. However, it is quite difficult to satisfy the numerous target requirements for biomedical nanomaterials based on metal NPs in the composition of dispersed oxides as well as textiles and/or biopolymers (“all in one”) to obtain a single universal multifunctional material that does not lose its properties during operation. It makes more sense to produce composites for purpose targeted applications, such as bactericidal and antiviral, hydrophobic coatings for laboratory surfaces, package and so on. Researches in this area are in progress.

Damage-reliability approach for fatigue crack propagation in MDPE gas pipe

Fatigue crack propagation tests are carried out on arc-shaped specimens prepared from MDPE gas pipes. Damage zone characterization is achieved using the diamond wafer sectioning technique from partially propagated and prematurely arrested cracks. Damage ahead of the crack-tip is used to assess a damage parameter and reliability based on statistical laws and subsequent use the PHIMECA Software. It is shown that a length and a width associated with a corresponding change in thickness at the fracture surface satisfactorily describe the damage zone size. The 3-parameter Weibull model gives the best reliability behavior and a critical lifetime of 82%. When considering separately both the Dugdale model and experimental damage zone measurements, it is possible to establish the evolution of the reliability index as a function of crack length. It is concluded that the reliability index approach based on damage provides a more consistent representation compared to analytical models.

Leveraging computer vision for adaptive learning in STEM education: effect of engagement and self-efficacy

In the field of Science, Technology, Engineering, and Mathematics (STEM) education, which aims to cultivate problem-solving skills, accurately assessing learners' engagement remains a significant challenge. We present a solution to this issue with the Real-time Automated STEM Engagement Detection System (RASEDS). This innovative system capitalizes on the power of artificial intelligence, computer vision, and the Interactive, Constructive, Active, and Passive (ICAP) framework. RASEDS uses You Only Learn One Representation (YOLOR) to detect and map learners' interactions onto the four levels of engagement delineated in the ICAP framework. This process informs the system's recommendation of adaptive learning materials, designed to boost both engagement and self-efficacy in STEM activities. Our study affirms that RASEDS accurately gauges engagement, and that the subsequent use of these adaptive materials significantly enhances both engagement and self-efficacy. Importantly, our research suggests a connection between elevated self-efficacy and increased engagement. As learners become more engaged in their learning process, their confidence is bolstered, thereby augmenting self-efficacy. We underscore the transformative potential of AI in facilitating adaptive learning in STEM education, highlighting the symbiotic relationship between engagement and self-efficacy.

Big advocacy, little recognition: the hidden work of Black patients in precision medicine

As cost-effective next-generation genome sequencing rapidly develops, calls for greater inclusion of Black people in genomic research, policy, and practice are necessary for effective translation of genomic science into precision population health and medicine. Employing a community-based participatory mixed methods research design, we developed a semi-structured survey that was disseminated to three cancer advocacy organizations. Of the 81 survey respondents 49 (60%) self-identified as Black, and 26 (32%) indicated a prior breast cancer diagnosis. Black participants’ expressed concerns about genetic testing were evenly distributed between concerns that could be addressed through genetic counseling (24%) and concerns about subsequent use of their genetic data (27%). Patient advocates contributed to contextualization of respondent concerns in terms of community experiences. Although genetic counseling services and policies governing genomic data use are not always accessible to many Black communities, advocates on our research team provided a bridge to discussion of the intersection between respondent concerns and the roles advocates play in filling gaps in access to genetic counseling and data governance. Concerns expressed by Black patients underscore a shared need among all patients for access to education, inclusion in research, and assurances regarding the use and handling of genetic data. Black cancer patients have joined in patient-led efforts to overcome systemic inequities in cancer care to improve their health outcomes through representation. Often their efforts are overshadowed by a relentless burden of continued health disparities. Future research should support their hidden work as a means to reduce barriers and improve representation in genomic databases.

Incorporating Signal Awareness in Source Code Modeling: An Application to Vulnerability Detection

AI models of code have made significant progress over the past few years. However, many models are actually not learning task-relevant source code features. Instead, they often fit non-relevant but correlated data, leading to a lack of robustness and generalizability, and limiting the subsequent practical use of such models. In this work, we focus on improving the model quality through signal awareness , i.e., learning the relevant signals in the input for making predictions. We do so by leveraging the heterogeneity of code samples in terms of their signal-to-noise content. We perform an end-to-end exploration of model signal awareness, comprising: (i) uncovering the reliance of AI models of code on task-irrelevant signals, via prediction-preserving input minimization; (ii) improving models’ signal awareness by incorporating the notion of code complexity during model training, via curriculum learning; (iii) improving models’ signal awareness by generating simplified signal-preserving programs and augmenting them to the training dataset; and (iv) presenting a novel interpretation of the model learning behavior from the perspective of the dataset, using its code complexity distribution. We propose a new metric to measure model signal awareness, Signal-aware Recall, which captures how much of the model’s performance is attributable to task-relevant signal learning. Using a software vulnerability detection use-case, our model probing approach uncovers a significant lack of signal awareness in the models, across three different neural network architectures and three datasets. Signal-aware Recall is observed to be in the sub-50s for models with traditional Recall in the high 90s, suggesting that the models are presumably picking up a lot of noise or dataset nuances while learning their logic. With our code-complexity-aware model learning enhancement techniques, we are able to assist the models toward more task-relevant learning, recording up-to 4.8× improvement in model signal awareness. Finally, we employ our model learning introspection approach to uncover the aspects of source code where the model is facing difficulty, and we analyze how our learning enhancement techniques alleviate it.

Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH

Antibody-fragment (Fab) therapy development has the potential to be accelerated by computational modelling and simulations that predict their target binding, stability, formulation, manufacturability, and the impact of further protein engineering. Such approaches are currently predicated on starting with good crystal structures that closely represent those found under the solution conditions to be simulated. A33 Fab, is an undeveloped immunotherapeutic antibody candidate that was targeted to the human A33 antigen homogeneously expressed in 95% cases of primary and metastatic colorectal cancers. It is now used as a very well characterised testing ground for developing analytics, formulation and protein engineering strategies, and to gain a deeper understanding of mechanisms of destabilisation, representative of the wider therapeutic Fab platform. In this article, we report the structure of A33 Fab in two different crystal forms obtained at acidic and basic pH. The structures overlapped with RMSD of 1.33 Å overall, yet only 0.5 Å and 0.76 Å for the variable- and constant regions alone. While most of the differences were within experimental error, the switch linker between the variable and the constant regions showed some small differences between the two pHs. The two structures then enabled a direct evaluation of the impact of initial crystal structure selection on the outcomes of molecular dynamics simulations under different conditions, and their subsequent use for determining best fit solution structures using previously obtained small-angle x-ray scattering (SAXS) data. The differences in the two structures did not have a major impact on MD simulations regardless of the pH, other than a slight persistence of structure affecting the solvent accessibility of one of the predicted APR regions of A33 Fab. Interestingly, despite being obtained at pH 4 and pH 9, the two crystal structures were more similar to the SAXS solution structures obtained at pH 7, than to those at pH 4 or pH 9. Furthermore, the P65 crystal structure from pH 4 was also a better representation of the solution structures at any other pH, than was the P1 structure obtained at pH 9. Thus, while obtained at different pH, the two crystal structures may represent highly (P65) and lesser (P1) populated species that both exist at pH 7 in solution. These results now lay the foundation for confident MD simulations of A33 Fab that rationalise or predict behaviours in a range of conditions.