Protection efforts have resulted in ~10% of existing fish biomass on coral reefs.

The amount of ocean protected from fishing and other human impacts has often been used as a metric of conservation progress. However, protection efforts have highly variable outcomes that depend on local conditions, which makes it difficult to quantify what coral reef protection efforts to date have actually achieved at a global scale. Here, we develop a predictive model of how local conditions influence conservation outcomes on ~2,600 coral reef sites across 44 ecoregions, which we used to quantify how much more fish biomass there is on coral reefs compared to a modeled scenario with no protection. Under the assumptions of our model, our study reveals that without existing protection efforts there would be ~10% less fish biomass on coral reefs. Thus, we estimate that coral reef protection efforts have led to approximately 1 in every 10 kg of existing fish biomass.

Single-cell Sequencing of Circulating Human Plasmablasts during Staphylococcus aureus Bacteremia.

Staphylococcus aureus is the major cause of healthcare-associated infections, including life-threatening conditions as bacteremia, endocarditis, and implant-associated infections. Despite adequate antibiotic treatment, the mortality of S. aureus bacteremia remains high. This calls for different strategies to treat this infection. In past years, sequencing of Ab repertoires from individuals previously exposed to a pathogen emerged as a successful method to discover novel therapeutic monoclonal Abs and understand circulating B cell diversity during infection. In this paper, we collected peripheral blood from 17 S. aureus bacteremia patients to study circulating plasmablast responses. Using single-cell transcriptome gene expression combined with sequencing of variable heavy and light Ig genes, we retrieved sequences from >400 plasmablasts revealing a high diversity with >300 unique variable heavy and light sequences. More than 200 variable sequences were synthesized to produce recombinant IgGs that were analyzed for binding to S. aureus whole bacterial cells. This revealed four novel monoclonal Abs that could specifically bind to the surface of S. aureus in the absence of Ig-binding surface SpA. Interestingly, three of four mAbs showed cross-reactivity with Staphylococcus epidermidis. Target identification revealed that the S. aureus-specific mAb BC153 targets wall teichoic acid, whereas cross-reactive mAbs BC019, BC020, and BC021 target lipoteichoic acid. All mAbs could induce Fc-dependent phagocytosis of staphylococci by human neutrophils. Altogether, we characterize the active B cell responses to S. aureus in infected patients and identify four functional mAbs against the S. aureus surface, of which three cross-react with S. epidermidis.

Determinants of CRISPR Cas12a nuclease activation by DNA and RNA targets.

The RNA-guided CRISPR-associated (Cas) enzyme Cas12a cleaves specific double-stranded (ds-) or single-stranded (ss-) DNA targets (in cis), unleashing non-specific ssDNA cleavage (in trans). Though this trans-activity is widely coopted for diagnostics, little is known about target determinants promoting optimal enzyme performance. Using quantitative kinetics, we show formation of activated nuclease proceeds via two steps whereby rapid binding of Cas12a ribonucleoprotein to target is followed by a slower allosteric transition. Activation does not require a canonical protospacer-adjacent motif (PAM), nor is utilization of such PAMs predictive of high trans-activity. We identify several target determinants that can profoundly impact activation times, including bases within the PAM (for ds- but not ssDNA targets) and sequences within and outside those complementary to the spacer, DNA topology, target length, presence of non-specific DNA, and ribose backbone itself, uncovering previously uncharacterized cleavage of and activation by RNA targets. The results provide insight into the mechanism of Cas12a activation, with direct implications on the role of Cas12a in bacterial immunity and for Cas-based diagnostics.

CD5L is a canonical component of circulatory IgM.

Immunoglobulin M (IgM) is an evolutionary conserved key component of humoral immunity, and the first antibody isotype to emerge during an immune response. IgM is a large (1 MDa), multimeric protein, for which both hexameric and pentameric structures have been described, the latter additionally containing a joining (J) chain. Using a combination of single-particle mass spectrometry and mass photometry, proteomics, and immunochemical assays, we here demonstrate that circulatory (serum) IgM exclusively exists as a complex of J-chain-containing pentamers covalently bound to the small (36 kDa) protein CD5 antigen-like (CD5L, also called apoptosis inhibitor of macrophage). In sharp contrast, secretory IgM in saliva and milk is principally devoid of CD5L. Unlike IgM itself, CD5L is not produced by B cells, implying that it associates with IgM in the extracellular space. We demonstrate that CD5L integration has functional implications, i.e., it diminishes IgM binding to two of its receptors, the FcαµR and the polymeric Immunoglobulin receptor. On the other hand, binding to FcµR as well as complement activation via C1q seem unaffected by CD5L integration. Taken together, we redefine the composition of circulatory IgM as a J-chain containing pentamer, always in complex with CD5L.

Diversification of refugia types needed to secure the future of coral reefs subject to climate change.

Identifying locations of refugia from the thermal stresses of climate change for coral reefs and better managing them is one of the key recommendations for climate change adaptation. We review and summarize approximately 30 years of applied research focused on identifying climate refugia to prioritize the conservation actions for coral reefs under rapid climate change. We found that currently proposed climate refugia and the locations predicted to avoid future coral losses are highly reliant on  excess heat metrics, such as degree heating weeks. However, many existing alternative environmental, ecological, and life-history variables could be used to identify other types of refugia that lead to the desired diversified portfolio for coral reef conservation. To improve conservation priorities for coral reefs, there is a need to evaluate and validate the predictions of climate refugia with long-term field data on coral abundance, diversity, and functioning. There is also the need to identify and safeguard locations displaying resistance toprolonged exposure to heat waves and the ability to recover quickly after thermal exposure. We recommend using more metrics to identify a portfolio of potential refugia sites for coral reefs that can avoid, resist, and recover from exposure to high ocean temperatures and the consequences of climate change, thereby shifting past efforts focused on avoidance to a diversified risk-spreading portfolio that can be used to improve strategic coral reef conservation in a rapidly warming climate.

Diversificación de los tipos de refugio necesarios para asegurar el futuro de los arrecifes de coral sujetos al cambio climático Resumen Una de las principales recomendaciones para la adaptación al cambio climático es identificar los refugios de los arrecifes de coral frente al estrés térmico del cambio climático y mejorar su gestión. Revisamos y resumimos ∼30 años de investigación aplicada centrada en la identificación de refugios climáticos para priorizar las acciones de conservación de los arrecifes de coral bajo un rápido cambio climático. Descubrimos que los refugios climáticos propuestos actualmente y las ubicaciones que pueden evitarlos dependen en gran medida de métricas de exceso de calor, como las semanas de calentamiento en grados (SCG). Sin embargo, existen muchas variables alternativas de historia vital, ambientales y ecológicas que podrían utilizarse para identificar otros tipos de refugios que resulten en el acervo diversificado que se desea para la conservación de los arrecifes de coral. Para mejorar las prioridades de conservación de los arrecifes de coral, es necesario evaluar y validar las predicciones sobre refugios climáticos con datos de campo a largo plazo sobre abundancia, diversidad y funcionamiento de los corales. También es necesario identificar y salvaguardar lugares que muestren resistencia a la exposición climática prolongada a olas de calor y la capacidad de recuperarse rápidamente tras la exposición térmica. Recomendamos utilizar más métricas para identificar un acervo de posibles lugares de refugio para los arrecifes de coral que puedan evitar, resistir y recuperarse de la exposición a las altas temperaturas oceánicas y las consecuencias del cambio climático, para así desplazar los esfuerzos pasados centrados en la evitación hacia un acervo diversificado de riesgos que pueda utilizarse para mejorar la conservación estratégica de los arrecifes de coral en un clima que se calienta rápidamente.

Outer membrane permeabilization by the membrane attack complex sensitizes Gram-negative bacteria to antimicrobial proteins in serum and phagocytes.

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.

Does Walking Help to Generate a Differential Diagnosis?

TheoryIn Medicine, arriving at the correct diagnosis is of paramount importance for patient health and safety, yet is a difficult task especially when a patient presents with symptoms that do not fit typical patterns of disease. This task can be further complicated by errors of judgment, with the failure to consider all possible diagnoses being the most common of such errors. In this study, we investigated the process of differential diagnosis generation within the growing evidence that diagnostic performance can be increased by activities such as walking as was previously shown in Oppezzo and Schwartz's 2014 study. Hypotheses: It was hypothesized that an increase in performance, as expressed by a greater number of plausible differential diagnoses, would be seen in the walking group. Method: Eighteen medical students in their last two months of pre-clerkship training and eighteen second year family medicine residents were shown four different lists of a constellation of signs and symptoms. Participants were asked to generate differential diagnoses over five minutes per each list. All participants sat when completing the first two lists (pretest phase), and then were equally and randomly assigned to sitting versus walking on a treadmill when completing the last two lists (post-test phase). The number of total and unique differential diagnoses generated was determined, before being submitted to a three-member expert panel who identified appropriate unique differential diagnoses. Results: Two-way mixed ANOVAs were conducted to investigate the impact of exercise on the number of total, unique, and appropriate unique ideas generated and compared between pretest and post-test phases. Conclusions: We conclude that there is neither an increase nor a decrease in the number or quality of differential diagnoses generated by the sitting and walking groups within a population that has acquired some level of expertise.

Does walking improve diagnosis of skin conditions at varying levels of medical expertise?

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.

Cerebrospinal fluid CXCL13 as a diagnostic marker of neuroborreliosis in children: a retrospective case-control study.

BACKGROUND: Lyme neuroborreliosis (LNB) is a frequent manifestation of Lyme disease in children and its current diagnosis has limitations. The elevation of the chemokine CXCL13 in the cerebrospinal fluid (CSF) of adult patients with LNB has been demonstrated and suggested as a new diagnostic marker. Our aim was to evaluate this marker in the CSF of children with suspected LNB and to determine a CXCL13 cut-off concentration that would discriminate between LNB and other central nervous system (CNS) infections. METHODS: For this single-center retrospective case-control study we used a diagnostic-approved ELISA to measure CXCL13 concentrations in the CSF of 185 children with LNB suspicion at presentation. Patients were classified into definite LNB (cases), non-LNB (controls with other CNS affections), and possible LNB. A receiver-operating characteristic curve was generated by comparison of cases and controls. RESULTS: CXCL13 was significantly elevated in the CSF of 53 children with definite LNB (median 774.7 pg/ml) compared to 91 control patients (median 4.5 pg/ml, p < 0.001). A cut-off of 55 pg/ml resulted in a sensitivity of 96.7% and a specificity of 98.1% for the diagnosis of definite LNB and the test exhibited a diagnostic odds ratio of 1525.3. Elevated CSF CXCL13 levels were also detected in three controls with viral meningitis (enterovirus n = 1, varicella-zoster virus n = 2) while other CNS affections such as idiopathic facial palsy did not lead to CXCL13 elevation. Of the 41 patients with possible LNB, 27% had CXCL13 values above the cut-off of 55 pg/ml (median 16.7 pg/ml). CONCLUSIONS: CSF CXCL13 is highly elevated in children during early LNB as previously shown in adults. CXCL13 is a highly sensitive and specific marker that helps to differentiate LNB from other CNS affections in children.

In vitro assessment of a collagen/alginate composite scaffold for regenerative endodontics.

AIM: To develop a biological scaffold that could be moulded to reproduce the geometry of a gutta-percha point with precision and allow the differentiation of mesenchymal stem cells into osteoblasts to be used as a regenerative endodontic material. METHODOLOGY: A collagen/alginate composite scaffold was cast into a sodium alginate mould to produce a gutta-percha point-like cone. Prior to gelation, the cone was seeded with human stem cells from the apical papilla (SCAPs) to evaluate cell/scaffold interactions. The reconstructed tissue was characterized after 8 days in culture. Elastic modulus, tissue compaction and cell differentiation were assessed. Student t-tests and the Mann-Whitney U test were performed. RESULTS: The fabrication method developed enabled the shape of a gutta-percha point to be mimicked with great accuracy and reproducibility (P = 0.31). Stem cells seeded into this composite scaffold were able to spread, survive and proliferate (P < 0.001). Moreover, they were able to differentiate into osteoblasts and produce calcified osseous extracellular matrix (P < 0.001). The construct showed no significant contraction after 8 days, preserving its shape and tip diameter (P = 0.58). CONCLUSIONS: The composite scaffold could present substantial benefits compared to synthetic materials. It could provide a favourable healing environment in the root canal conducive for regenerative endodontics and is therefore appropriate to be evaluated in vivo in further studies.

Non-cognitive selected students do not outperform lottery-admitted students in the pre-clinical stage of medical school.

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.

The development of self-regulated learning during the pre-clinical stage of medical school: a comparison between a lecture-based and a problem-based curriculum.

Society expects physicians to always improve their competencies and to be up to date with developments in their field. Therefore, an important aim of medical schools is to educate future medical doctors to become self-regulated, lifelong learners. However, it is unclear if medical students become better self-regulated learners during the pre-clinical stage of medical school, and whether students develop self-regulated learning skills differently, dependent on the educational approach of their medical school. In a cross-sectional design, we investigated the development of 384 medical students' self-regulated learning skills with the use of the Self-Regulation of Learning Self-Report Scale. Next, we compared this development in students who enrolled in two distinct medical curricula: a problem-based curriculum and a lectured-based curriculum. Analysis showed that more skills decreased than increased during the pre-clinical stage of medical school, and that the difference between the curricula was mainly caused by a decrease in the skill evaluation in the lecture-based curriculum. These findings seem to suggest that, irrespective of the curriculum, self-regulated learning skills do not develop during medical school.

Self-regulated learning and academic performance in medical education.

CONTENT: Medical schools aim to graduate medical doctors who are able to self-regulate their learning. It is therefore important to investigate whether medical students' self-regulated learning skills change during medical school. In addition, since these skills are expected to be helpful to learn more effectively, it is of interest to investigate whether these skills are related to academic performance. METHODS: In a cross-sectional design, the Self-Regulation of Learning Self-Report Scale (SRL-SRS) was used to investigate the change in students' self-regulated learning skills. First and third-year students (N = 949, 81.7%) SRL-SRS scores were compared with ANOVA. The relation with academic performance was investigated with multinomial regression analysis. RESULTS: Only one of the six skills, reflection, significantly, but positively, changed during medical school. In addition, a small, but positive relation of monitoring, reflection, and effort with first-year GPA was found, while only effort was related to third-year GPA. CONCLUSIONS: The change in self-regulated learning skills is minor as only the level of reflection differs between the first and third year. In addition, the relation between self-regulated learning skills and academic performance is limited. Medical schools are therefore encouraged to re-examine the curriculum and methods they use to enhance their students' self-regulated learning skills. Future research is required to understand the limited impact on performance.

CaCO3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma.

MicroRNA (miR)-200c suppresses the initiation and progression of oral squamous cell carcinoma (OSCC), the most prevalent head and neck cancer with high recurrence, metastasis, and mortality rates. However, miR-200c-based gene therapy to inhibit OSCC growth has yet to be reported. To develop an miR-based gene therapy to improve the outcomes of OSCC treatment, this study investigates the feasibility of plasmid DNA (pDNA) encoding miR-200c delivered via nonviral CaCO3-based nanoparticles to inhibit OSCC tumor growth. CaCO3-based nanoparticles with various ratios of CaCO3 and protamine sulfate (PS) were used to transfect pDNA encoding miR-200c into OSCC cells, and the efficiency of these nanoparticles was evaluated. The proliferation, migration, and associated oncogene production, as well as in vivo tumor growth for OSCC cells overexpressing miR-200c, were also quantified. It was observed that, while CaCO3-based nanoparticles improve transfection efficiencies of pDNA miR-200c, the ratio of CaCO3 to PS significantly influences the transfection efficiency. Overexpression of miR-200c significantly reduced proliferation, migration, and oncogene expression of OSCC cells, as well as the tumor size of cell line-derived xenografts (CDX) in mice. In addition, a local administration of pDNA miR-200c using CaCO3 delivery significantly enhanced miR-200c transfection and suppressed tumor growth of CDX in mice. These results strongly indicate that the nanocomplexes of CaCO3/pDNA miR-200c may potentially be used to reduce oral cancer recurrence and improve clinical outcomes in OSCC treatment, while more comprehensive examinations to confirm the safety and efficacy of the CaCO3/pDNA miR-200c system using various preclinical models are needed.

Agnostic B cell selection approach identifies antibodies against K. pneumoniae that synergistically drive complement activation.

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.

Students' self-explanations while solving unfamiliar cases: the role of biomedical knowledge.

OBJECTIVE: General guidelines for teaching clinical reasoning have received much attention, despite a paucity of instructional approaches with demonstrated effectiveness. As suggested in a recent experimental study, self-explanation while solving clinical cases may be an effective strategy to foster reasoning in clinical clerks dealing with less familiar cases. However, the mechanisms that mediate this benefit have not been specifically investigated. The aim of this study was to explore the types of knowledge used by students when solving familiar and less familiar clinical cases with self-explanation. METHODS: In a previous study, 36 third-year medical students diagnosed familiar and less familiar clinical cases either by engaging in self-explanation or not. Based on an analysis of previously collected data, the present study compared the content of self-explanation protocols generated by seven randomly selected students while solving four familiar and four less familiar cases. In total, 56 verbal protocols (28 familiar and 28 less familiar) were segmented and coded using the following categories: paraphrases, biomedical inferences, clinical inferences, monitoring statements and errors. RESULTS: Students provided more self-explanation segments from less familiar cases (M = 275.29) than from familiar cases (M = 248.71, p = 0.046). They provided significantly more paraphrases (p = 0.001) and made more errors (p = 0.008). A significant interaction was found between familiarity and the type of inferences (biomedical versus clinical, p = 0.016). When self-explaining less familiar cases, students provided significantly more biomedical inferences than familiar cases. CONCLUSIONS: Lack of familiarity with a case seems to stimulate medical students to engage in more extensive thinking during self-explanation. Less familiar cases seem to activate students' biomedical knowledge, which in turn helps them to create new links between biomedical and clinical knowledge, and eventually construct a more coherent mental representation of diseases. This may clarify the previously found positive effect that self-explanation has on the diagnosis of unfamiliar cases.

To observe or not to observe peers when learning physical examination skills; that is the question.

BACKGROUND: Learning physical examination skills is an essential element of medical education. Teaching strategies include practicing the skills either alone or in-group. It is unclear whether students benefit more from training these skills individually or in a group, as the latter allows them to observing their peers. The present study, conducted in a naturalistic setting, investigated the effects of peer observation on mastering psychomotor skills necessary for physical examination. METHODS: The study included 185 2nd-year medical students, participating in a regular head-to-toe physical examination learning activity. Students were assigned either to a single-student condition (n = 65), in which participants practiced alone with a patient instructor, or to a multiple-student condition (n = 120), in which participants practiced in triads under patient instructor supervision. The students subsequently carried out a complete examination that was videotaped and subsequently evaluated. Student's performance was used as a measure of learning. RESULTS: Students in the multiple-student condition learned more than those who practiced alone (81% vs 76%, p < 0.004). This result possibly derived from a positive effect of observing peers; students who had the possibility to observe a peer (the second and third students in the groups) performed better than students who did not have this possibility (84% vs 76%, p <. 001). There was no advantage of observing more than one peer (83.7% vs 84.1%, p > .05). CONCLUSIONS: The opportunity to observe a peer during practice seemed to improve the acquisition of physical examination skills. By using small groups instead of individual training to teach physical examination skills, health sciences educational programs may provide students with opportunities to improve their performance by learning from their peers through modelling.

MEMO1 Is Required for Ameloblast Maturation and Functional Enamel Formation.

Coordinated mineralization of soft tissue is central to organismal form and function, while dysregulated mineralization underlies several human pathologies. Oral epithelial-derived ameloblasts are polarized, secretory cells responsible for generating enamel, the most mineralized substance in the human body. Defects in ameloblast development result in enamel anomalies, including amelogenesis imperfecta. Identifying proteins critical in ameloblast development can provide insight into specific pathologies associated with enamel-related disorders or, more broadly, mechanisms of mineralization. Previous studies identified a role for MEMO1 in bone mineralization; however, whether MEMO1 functions in the generation of additional mineralized structures remains unknown. Here, we identify a critical role for MEMO1 in enamel mineralization. First, we show that Memo1 is expressed in ameloblasts and, second, that its conditional deletion from ameloblasts results in enamel defects, characterized by a decline in mineral density and tooth integrity. Histology revealed that the mineralization defects in Memo1 mutant ameloblasts correlated with a disruption in ameloblast morphology. Finally, molecular profiling of ameloblasts and their progenitors in Memo1 oral epithelial mutants revealed a disruption to cytoskeletal-associated genes and a reduction in late-stage ameloblast markers, relative to controls. Collectively, our findings integrate MEMO1 into an emerging network of molecules important for ameloblast development and provide a system to further interrogate the relationship of cytoskeletal and amelogenesis-related defects.

Purified complement C3b triggers phagocytosis and activation of human neutrophils via complement receptor 1.

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.

Artificial surface labelling of Escherichia coli with StrepTagII antigen to study how monoclonal antibodies drive complement-mediated killing.

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.