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Abstract Antibody-dependent complement activation plays a key role in the natural human immune response to infections. Currently, the understanding of which antibody-antigen combinations drive a potent complement response on bacteria is limited. Here, we develop an antigen-agnostic approach to stain and single-cell sort human IgG memory B cells recognizing intact bacterial cells, keeping surface antigens in their natural context. With this method we successfully identified 29 antibodies against K. pneumoniae, a dominant cause of hospital-acquired infections with increasing antibiotic resistance. Combining genetic tools and functional analyses, we reveal that the capacity of antibodies to activate complement on K. pneumoniae critically depends on their antigenic target. Furthermore, we find that antibody combinations can synergistically activate complement on K. pneumoniae by strengthening each other’s binding in an Fc-independent manner. Understanding the molecular basis of effective complement activation by antibody combinations to mimic a polyclonal response could accelerate the development of antibody-based therapies against problematic infections.

Abstract Antibodies play a key role in the immune defence against Gram-negative bacteria. After binding to bacterial surface antigens, IgG and IgM can activate the complement system and trigger formation of lytic membrane attack complex (MAC) pores. Molecular studies to compare functional activity of antibodies on bacteria are hampered by the limited availability of well-defined antibodies against bacterial surface antigens. Therefore, we genetically engineered E. coli by expressing the StrepTagII antigen into outer membrane protein X (OmpX) and validated that these engineered bacteria were recognised by anti-StrepTagII antibodies. We then combined this antigen–antibody system with a purified complement assay to avoid interference of serum components and directly compare MAC-mediated bacterial killing via IgG1 and pentameric IgM. While both IgG1 and IgM could induce MAC-mediated killing, we show that IgM has an increased capacity to induce complement-mediated killing of E. coli compared to IgG1. While Fc mutations that enhance IgG clustering after target binding could not improve MAC formation, mutations that cause formation of pre-assembled IgG hexamers enhanced the complement activating capacity of IgG1. Altogether, we here present a system to study antibody-dependent complement activation on E. coli and show IgM’s enhanced capacity over IgG to induce complement-mediated lysis of E. coli.

Abstract The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.

Summary: Bacterial CRISPR systems provide acquired immunity against invading nucleic acids by activating RNA-programmable RNases and DNases. Cas13a and Cas12a enzymes bound to CRISPR RNA (crRNA) recognize specific nucleic acid targets, initiating cleavage of the targets as well as non-target (trans) nucleic acids. Here, we examine the kinetics of single-turnover target and multi-turnover trans-nuclease activities of both enzymes. High-turnover, non-specific Cas13a trans-RNase activity is coupled to rapid binding of target RNA. By contrast, low-turnover Cas12a trans-nuclease activity is coupled to relatively slow cleavage of target DNA, selective for DNA over RNA, indifferent to base identity, and preferential for single-stranded substrates. Combining multiple crRNA increases detection sensitivity of targets, an approach we use to quantify pathogen DNA in samples from patients suspected of Buruli ulcer disease. Results reveal that these enzymes are kinetically adapted to play distinct roles in bacterial adaptive immunity and show how kinetic analysis can be applied to CRISPR-based diagnostics.

Infections with Gram-negative bacteria form an increasing risk for human health due to antibiotic resistance. Our immune system contains various antimicrobial proteins that can degrade the bacterial cell envelope. However, many of these proteins do not function on Gram-negative bacteria, because the impermeable outer membrane of these bacteria prevents such components from reaching their targets. Here we show that complement-dependent formation of Membrane Attack Complex (MAC) pores permeabilizes this barrier, allowing antimicrobial proteins to cross the outer membrane and exert their antimicrobial function. Specifically, we demonstrate that MAC-dependent outer membrane damage enables human lysozyme to degrade the cell wall of E. coli. Using flow cytometry and confocal microscopy, we show that the combination of MAC pores and lysozyme triggers effective E. coli cell wall degradation in human serum, thereby altering the bacterial cell morphology from rod-shaped to spherical. Completely assembled MAC pores are required to sensitize E. coli to the antimicrobial actions of lysozyme and other immune factors, such as Human Group IIA-secreted Phospholipase A2. Next to these effects in a serum environment, we observed that the MAC also sensitizes E. coli to more efficient degradation and killing inside human neutrophils. Altogether, this study serves as a proof of principle on how different players of the human immune system can work together to degrade the complex cell envelope of Gram-negative bacteria. This knowledge may facilitate the development of new antimicrobials that could stimulate or work synergistically with the immune system.

In the present study, we compared the mindset of preuniversity students and (primary and secondary) vocational students. Participants comprised of 173 students attending preuniversity education and 101 students attending vocational education. All participants completed a mindset questionnaire. We expected, based on previous educational experiences, that preuniversity students would show on average a higher score (i.e., more of a growth mindset) than vocational students. Results indicated, however, that there was hardly any difference in mindset between vocational and preuniversity students. The mindset of adolescents is therefore not influenced by the level of education.

The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.

Student-teacher relationships in higher education take place in two embedded social contexts: the in-class environment and the educational institute in which the in-class environment is situated. The interplay between the two contexts and their association with students' motivation was studied in the current study. In a broad sample (N=597), perceptions of student-staff relationship quality, need-supportive teaching in class, and autonomous and controlled motivation were measured. Perceptions of in-class teacher involvement were associated with autonomous motivation, while perceptions of in-class teacher structure were associated with controlled motivation. The association of perceptions of in-class teacher involvement and autonomy support with autonomous motivation seemed to be suppressed by perceptions of trust in benevolence. This indicates that students can get a 'just tell me what to do'-attitude when trust in benevolence makes them shift from being supported to depending on support. Studying both embedded contexts in one analysis, resulted in a more fine-grained insight.

In the present study, we compared the mindset of preuniversity students and (primary and secondary) vocational students. Participants comprised of 173 students attending preuniversity education and 101 students attending vocational education. All participants completed a mindset questionnaire. We expected, based on previous educational experiences, that preuniversity students would show on average a higher score (i.e., more of a growth mindset) than vocational students. Results indicated, however, that there was hardly any difference in mindset between vocational and preuniversity students. The mindset of adolescents is therefore not influenced by the level of education.

Supportive relationships between students and their educational faculty and staff can foster positive outcomes such as students' involvement and development. However, research investigating how students perceive the quality of their relationships with educational faculty/staff (i.e., relationship quality) so far remains scarce. This study's aim was to gain more insight into the construct of relationship quality in higher education using a qualitative approach. Students' descriptions of their positive (n = 294) and negative relationship experiences (n = 395) were collected using a critical incident technique (final sample N = 513 critical incidents) followed by a template analysis with a priori themes (i.e., relationship quality dimensions: trust in honesty, trust in benevolence, satisfaction, affective commitment, affective conflict). Results indicated that students most often mentioned trust in honesty and trust in benevolence. Affective conflict was not always explicitly mentioned in negative experiences, nor satisfaction in positive experiences. Descriptions of trust in benevolence (n = 355) were equally distributed over positive and negative incidents. However, trust in honesty was more often referred to in negative (n = 145) than in positive incidents (n = 51). The results indicated that students considered timely response to assignments and emails important, and teachers showing interest and attention. The study's findings provide a new view of how students might positively and negatively perceive the quality of their relationship with educational faculty and staff. This study adds to the theoretical and practical implications of relationship quality research in higher education and how relational aspects are important for students.

Background: To date, studies that have investigated the bonds between students and their institution have emphasized the importance of student-staff relationships. Measuring the quality of those relationships (i.e., relationship quality) appears to help with investigating the relational ties students have with their higher education institutions. Growing interest has arisen in further investigating relationship quality in higher education, as it might predict students' involvement with the institution (e.g., student engagement and student loyalty). So far, most studies have used a cross-sectional design, so that causality could not be determined. Aims: The aim of this longitudinal study was twofold. First, we investigated the temporal ordering of the relation between the relationship quality dimensions of trust (in benevolence and honesty) and affect (satisfaction, affective commitment, and affective conflict). Second, we examined the ordering of the paths between relationship quality, student engagement, and student loyalty. Our objectives were to gain a deeper understanding of the relationship quality construct in higher education and its later outcomes. Sample: Participants (N = 1649) were students from three Dutch higher education institutions who were studying in a technology economics or social sciences program. Methods: Longitudinal data from two time points were used to evaluate two types of cross-lagged panel models. In the first analysis, we could not assume measurement invariance for affective conflict over time. Therefore, we tested an alternative model without affective conflict, using the latent variables of trust and affect, the student engagement dimensions and student loyalty. In the second type of model, we investigated the manifest variables of relationship quality, student engagement, and student loyalty. The hypotheses were tested by evaluating simultaneous comparisons between estimates. Results: Results indicated that the relation between relationship quality at Time 1 with student engagement and loyalty at Time 2 was stronger than the reverse ordering in the first model. In the second model, results indicated that cross-lagged relations between trust in benevolence and trust in honesty at Time 1 and affective commitment, affective conflict, and satisfaction at Time 2 were more likely than the reverse ordering. Furthermore, cross-lagged relations from relationship quality at Time 1 to student engagement and student loyalty at Time 2 also supported our hypothesis. Conclusions: This study contributes to the existing higher education literature, indicating that students' trust in the quality of their relationship with faculty/staff is essential for developing students' affective commitment and satisfaction and for avoiding conflict over time. Second, relationship quality factors positively influence students' engagement in their studies and their loyalty towards the institution. A relational approach to establishing (long-lasting) bonds with students appears to be fruitful as an approach for educational psychologists and for practitioners' guidance and strategies. Recommendations are made for future research to further examine relationship quality in higher education in Europe and beyond.

Bacterial CRISPR systems provide acquired immunity against invading nucleic acids by activating RNA-programmable RNases and DNases. Cas13a and Cas12a enzymes bound to CRISPR RNA (crRNA) recognize specific nucleic acid targets, initiating cleavage of the targets as well as non-target (trans) nucleic acids. Here, we examine the kinetics of single-turnover target and multi-turnover trans-nuclease activities of both enzymes. High-turnover, non-specific Cas13a trans-RNase activity is coupled to rapid binding of target RNA. By contrast, low-turnover Cas12a trans-nuclease activity is coupled to relatively slow cleavage of target DNA, selective for DNA over RNA, indifferent to base identity, and preferential for single-stranded substrates. Combining multiple crRNA increases detection sensitivity of targets, an approach we use to quantify pathogen DNA in samples from patients suspected of Buruli ulcer disease. Results reveal that these enzymes are kinetically adapted to play distinct roles in bacterial adaptive immunity and show how kinetic analysis can be applied to CRISPR-based diagnostics.

The use of walking workstations in educational and work settings has been shown to improve cognitive abilities. At the same time, it has been repeatedly shown that medical residents around the world do not meet exercise guidelines, mainly due to a scarcity of available free time. Our study investigates the boundaries of the previously observed phenomenon of improved cognitive performance with physical activity using materials that represent real life tasks. Participants had different level of expertise and involved second year psychology students, medical students, and family medicine residents. We examined the effect of being physically inactive (i.e., sitting) or active (i.e., walking) while diagnosing multiple complex presentations of four skin conditions. We assumed that being physically active, irrespective of the level of expertise, will bolster diagnostic performance. Our findings show, however, that being physically active does not change the performance level of participants with different levels of medical expertise. Implications for medical education and suggestions for further research will be discussed.

When compared to high performers, low performers generally have more difficulty to accurately estimate their own performance. This has been explained by low performers being both unskilled and unaware about their performance. However, Miller and Geraci "Journal of Experimental Psychology: Learning, Memory, and Cognition," 37(2), 502-506, (2011) found that low performing university students also assigned less confidence to their estimates (i.e., second-order judgments, SOJs), indicating some metacognitive awareness of their poor calibration. The current study examined whether the relationship between calibration accuracy and confidence in performance estimates is more general, and exists irrespective of performance level, not only for university students but also for secondary school students. We asked university students and secondary school students to estimate their exam grade after taking their exam, and to provide a second-order judgement). The results showed that for university students, poor calibration accuracy was indeed accompanied by low confidence scores, independent from performance level. For secondary school students however, calibration accuracy was unrelated to confidence scores, suggesting a less developed metacognitive awareness.

Grades provide students with information about their level of performance. However, grades may also make students more aware of how well they have estimated their performance, their so-called calibration accuracy. This longitudinal quasi-experimental study, set in secondary education, examined how to increase students' awareness of the accuracy of their grade estimates in order to improve their calibration accuracy. During an entire school year, students from year 1, 2, and 3 provided grade estimates after each of their French exams. Subsequently, when students received their grades, the level of reflection support on their earlier estimates was manipulated. The first group of students just received their grade, the second group had to calculate the difference between their estimate and the actual grade, and the third group also had to reflect on reasons for a possible mismatch. We expected that more reflection support would lead to more improvement in calibration accuracy. Results showed that providing grade estimates already improved calibration accuracy over the school year, regardless of level of reflection support. This finding shows that asking for grade estimates is an easy-to-implement way to improve calibration accuracy of students in secondary education.

Mindset plays a pivotal role in academic achievement. In particular, a growth mindset is related to academic success. This study explored the role of mindset in Secondary Vocational Education and Training (VET). Participants were 1005 VET students attending eight different vocational programmes on three qualification levels in the rural southwest of the Netherlands. They filled out an online questionnaire that was combined with the school administration system for demographical information and school results. Results showed that 13.9% of the participants had a fixed mindset, 47.3% a growth mindset, and 38.8% a mixed mindset. Our findings indicate that VET students' mean mindset does not substantially differ from the mindset of students in other forms of education. However, the majority of VET students does not have a growth mindset and mindset and academic achievement seem to be unrelated.

The present study investigates the effects of different kinds of praise on 108 students in vocational education, using a similar design as the original mindset studies. Students worked on a set of Raven's Standard Progressive Matrices and either received praise for effort, received praise for intelligence, or were in the control group. Results were not in line with mindset theory. We expected differences in goal choice and performance after experiencing setbacks between students who were praised for effort or who were praised for intelligence, but both groups reacted in the same way. Our results are in line with previous studies that also did not succeed in finding a relation between mindset and academic performance. This study shows that even when the original procedure used in Mueller and Dweck's experiments was followed, vocational education students were not influenced by the type of praise (i.e. mindset) to which they were exposed.

The effectiveness of a mindset intervention in secondary vocational education and training was investigated. Students (n = 55) enrolled in a mathematics course participated in an online mindset intervention for a period of 10 weeks. Both pretest and posttest scores on mindset and mathematics results were compared with students who did not get a mindset intervention. We expected that students in the intervention group will show a growth in mindset and therefore have better results on the mathematics test at the end of the course compared to students in the control group. This effect will be stronger for lower performing students and students with a fixed mindset. Results showed that at the end of this period, the scores on the mathematics test were higher for both groups, but that there were no significant differences between groups. Furthermore, there was no effect of the intervention on the mindset of the students and there was no significant relation between mindset and scores on mathematics. Overall, our findings are in line with an increasing body of studies that did not find a relation between mindset and academic achievement.

Alumni can be of enormous value for higher education institutions because of the time and money they can spend on their former institution. Going beyond prior research that has as yet mostly considered alumni giving, this study focuses on exploring drivers for non-monetary alumni behavior (i.e., alumni loyalty). Modeling analysis was conducted on 152 alumni responses from two Dutch universities of applied sciences. Based on social exchange theory, a structural equation model was tested in which relationship quality dimensions were associated with student engagement, which in turn were related to alumni loyalty. Findings showed that the relationship quality dimensions of trust in benevolence and affective commitment had a statistically significant positive association with the student engagement dimensions absorption, dedication and vigor, and with alumni loyalty. The findings of this study support the importance of relationship quality dimensions in higher education for initiating long-lasting relationships with students even after their graduation, and for establishing non-monetary contributions in terms of alumni loyalty.

This experimental study explores whether feedback in the form of standards helps students in giving more accurate performance estimates not only on current tasks but also on new, similar tasks and whether performance level influences the effect of standards. We provided 122 first-year psychology students with seven texts that contained key terms. After reading each text, participants recalled the correct definitions of the key terms and estimated the quality of their recall. Half of the participants subsequently received standards and again estimated their own performance. Results showed that providing standards led to better calibration accuracy, both on current tasks and on new, similar tasks, when standards were not available yet. Furthermore, with or without standards, high performers calibrated better than low performers. However, results showed that especially low performers' calibration accuracy benefitted from receiving standards.

We study the Einstein-Yang-Mills-Higgs (EYMH) system with a positive cosmological constant in the Bianchi type I space-time with locally rotational symmetry (LRS). In particular, we consider the nonlinear interaction of the Higgs field with the Yang-Mills field coupled to an unknown gravitational field. For the considered model, from certain additional conditions (the temporal gauge and some symmetries), we derive the conservation laws for the field equations and we then deduce the exact formulation of equations in the geometric framework. Furthermore, using an iterative approch and some mathematical analysis tools, we study the above system of equations. We then establish a global existence result for the homogeneous solution and we analyse its asymptotic behaviour. [ABSTRACT FROM AUTHOR]

This study investigated the overall "quality" of the interpersonal relationship students have with faculty and staff, that is, relationship quality (RQ). In relationship management research, RQ is paramount for the creation of bonds with customers, which in turn is necessary for the sustainability of organizations, that is, continuity and growth. In higher education, it is not only recent changes in funding of education that urge us to further investigate RQ, as students having relational bonds with their teachers and faculty/staff is important as well. These relationships are expected to positively influence students' college experiences. Although educational literature addresses the importance of student-faculty relationships, little is known about students' perceptions of the quality of their relationship with their program. The aim of this study was therefore to get a more in-depth understanding of the concept and measurement of RQ within a higher education context. To that end, an existing RQ scale was used measuring five dimensions: trust in honesty, trust in benevolence, satisfaction, affective commitment, and affective conflict. A confirmatory factor analysis (CFA) was conducted on survey responses of 551 students from a Dutch university of applied sciences. Next to the CFA, a small-scale focus group discussion was held to validate the quantitative findings of students' perceptions on RQ. The findings confirm that the RQ instrument is an adequate instrument to investigate RQ in a higher education context. Additional qualitative findings also suggest that students acknowledge the relevance of RQ and the need for having a good relationship with their faculty and staff.

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Medical schools all over the world select applicants using non-cognitive and cognitive criteria. The predictive value of these different types of selection criteria has however never been investigated within the same curriculum while using a control group. We therefore set up a study that enabled us to compare the academic performance of three different admission groups, all composed of school-leaver entry students, and all enrolled in the same Bachelor curriculum: students selected on non-cognitive criteria, students selected on cognitive criteria and students admitted by lottery. First-year GPA and number of course credits (ECTS) at 52 weeks after enrollment of non-cognitive selected students (N = 102), cognitive selected students (N = 92) and lottery-admitted students (N = 356) were analyzed. In addition, chances of dropping out, probability of passing the third-year OSCE, and completing the Bachelor program in 3 years were compared. Although there were no significant differences between the admission groups in first-year GPA, cognitive selected students had obtained significantly more ECTS at 52 weeks and dropped out less often than lottery-admitted students. Probabilities of passing the OSCE and completing the bachelor program in 3 years did not significantly differ between the groups. These findings indicate that the use of only non-cognitive selection criteria is not sufficient to select the best academically performing students, most probably because a minimal cognitive basis is needed to succeed in medical school.